Mechanisms behind interplant communication - ‘how do plants talk?’

TOWARDS AN INTEGRATION OF THE MECHANISMS OF INTERPLANT COMMUNICATION

MASTER THESIS Veronika Ramovs Mentor: Prof. Dr. Corné Pieterse

Introduction 2

Abstract When plants are under or pathogen attack, or experiencing abiotic stress conditions, they can emit signalling molecules that can be received and perceived by their neighbouring plants. Receiving plants can respond to such signals by inducing defence responses, changing growth or priming their defences for possible future attack. Such transfer of information between plants, also named ‘interplant communication’, can take place above or below ground. Above ground signalling molecules are transported through air as blends of volatile compounds of specific concentrations. Below ground, plants can send and perceive signals through roots or common mycorrhizal networks. Numerous studies performed over the last three decades brought better understanding of the identity of signaling molecules and mechanisms behind the emission and perception of them.

However there is still a lack of information, especially on the molecular mechanisms of signalling pathways involved in the signal emission, perception and processing. Furthermore, most of the published studies have so far focused on the specific types of signalling taking place either above or below ground, considering plant tissues as independent systems. This thesis presents review of all currently known mechanisms of interplant communication, the circumstances under which these mecahnsims are triggered and the signal molecules that are involved. Different mechanisms of interplant communication are discussed in integrated manner, taking into account plants as whole organisms that are able to perceive and simultanesously respond to several stimuli.

Table of contents

I. INTRODUCTION II. AIRBORNE VOLATILE SIGNALLING AS MECHANISM OF INTERPLANT COMMUNICATION A. Starting the conversation A.1. Cues for interplant signalling, their recognition and signalling pathways leading to VOCs synthesis A.2. Identity of signalling molecules A.3. Release of VOCs B. Receiving and responding to the message III. MECHANISMS OF UNDERGROUND INTERPLANT COMMUNICATION A. Signalling through roots B. Signalling through underground common mycorrhizal network (CMN) IV. PLANTS AS WHOLE ORGANISMS – INTEGRATED VIEW ON THE INTERPLANT COMMUNICATION V. CONCLUSIONS VI. REFERENCES

Introduction 1

Introduction another. In this thesis I first review all currently known mechanisms of interplant communication, The ability of plants to emit and intercept signalling the circumstances under which these mecahnsims molecules from other plants has been one of the are triggered and the signal molecules that are developing research topics in molecular and plant involved. This is essential to establish where the data ecology over the last three decades. Ever since is lacking, which questions still need to be answered 1 2 Baldwin and Schultz as well as Rhoades provided and how to proceed. Secondly, I compare and first indications of interplant communication, discuss currently known data in the integrated 3 questions about community evolution , manner, placing different types of interplant circumstances under which plants exhibit such communication in the same context and trying to behaviour and questions on physiological and discuss their impact on each other, taking into molecular mechanisms behind it arose. With more account plants as whole organisms that are able to and more evidence that plants are able to perceive perceive and simultanesously respond to several and respond to the signals released from stimuli. neighbouring plants also in the natural environment4–8, interplant communication is Volatile signalling as mechanism becoming a widely recognised and accepted phenomena. Many of its aspects however remain of interplant communication somewhat controversial. It is still debated whether With more than 1700 currently known compounds11, plants deliberately send messages to their volatiles play an important role in the mechanisms of neighbours, or whether interplant communication is plant interaction within the environment. They are a side effect of other functions of signalling part of plant defence and reproductive mechanisms, molecules. Furthermore, it is unknown what this involving interactions of plants with organisms of means from an evolutionary perspective9. Current higher trophic levels (Fig. 1). The first implications of general opinions lean toward the idea of plants interplant communication, in which herbivore ‘eavesdropping’ on volatile organic compounds resistance was shown to increase in plants growing (VOCs) of their neighbours and consequently in close range to herbivore-attacked plants1,2, was responding by adapting their phenotypes and suggested to be mediated through air-borne therefore enhancing their fitness. Supporting such a signals12. These pioneer findings caused mixed conceptfurther is also recently elucidated within- responses in public and the topic was popularly plant signalling through aerial space, mediated by named ‘talking trees’. similar compounds as found to induce response in Since then, new evidence has been gathered under neighbouring plants10. laboratory, as well as field conditions. This evidence At the same time there are also an increasing has confirmed that plants respond to volatile number of reports on the different mechanisms of airborne molecules emitted from other plants and interplant communication. Besides well established identified to some extend identity of molecules and airborne communication other mechanisms of mechanisms of action involved. The emission and interplant communication have been observed perception of VOCs are currently one of the most below ground. Modern approaches and techniques researched and acknowledged types of interplant are enabling better understanding of the identity of communication. signalling molecules, as well as molecular mechanisms leading to their release, their STARTING THE CONVERSATION perception and formation of the response by the receiving plant. Wide transcriptional analyses can be Cues for interplant signalling, their used to connect biochemical, ecological and recognition and signalling pathways physiological data within a genetic framework. leading to VOCs synthesis Most of the published data on interplant What are the cues causing plants to release VOCs communication has so far focused on specific types whichare able to affect their neighbouring plants of signalling taking place either above or below and what is the mechanism behind their perception ground. However, since plant tissues do not act as and recognition? As sessile organisms, plants had to independent organisms, an integrated approach is develop diverse and numerous defensive needed to fully understand how plants signal to one Volatile signalling as mechanism of interplant communication 1

Fig. 1: Roles of the plant volatile organic compounds (VOCs) in interaction of plants within the environment. VOCs play part in defence responses, such as a ‘cry for help’ and protection under abiotic or biotic stress conditions, transfer of information within and between plants and in reproductive mechanisms, such as attraction of pollinators and seed dispersal.

mechanisms in order to survive constant attacks by Several experiments, where lima bean plants and pathogens. Therefore it is not very (Phaseolus lunatus) were infestated with spider surprising that most plants shown to be emitters of mites (Tetranychus urticae, Acari), showed that airborne VOCs involved in the interplant receiving lima beans responded to volatile communication are responding to damage caused by compounds from attacked plants13–15. HIPVs released attackers. from lima bean plants that were infested with the leaf miners caused a defence response in exposed RESPONSE TO HERBIVORY 16 Arabidopsis thaliana plants . Damage caused by Herbivore feeding has been known to invoke several species of beetles (Coleoptera) was also defence responses in the neighbours of attacked shown to be able to induce release of HIPVs that plants since the first observations of interplant resulted in defence responses in receiving plants; communication. In 1983 David F. Rhoades, one of cereal leaf beetle (Oelema melanipus) in the the pioneers in the field of plant-plant signaling, common wheat (Triticumae stivum), common oat reported that Sitka willow (Salix sithensis) trees (Avena sativa) in barley plants (Hordeum vulgare)17 respond to their neighbouring willow trees, attacked and Gynandrobrotica guerreroensis and Cerotoma by tent caterpillars (Malacosomacalifornicum- ruficornis beetles in the lima bean plants10. Other pluviale). Because no root connections between herbivorous arthropods, reported to be involved in trees were found, Rhoades assumed that interplant communication, include aphids neighbouring trees are able to sense airborne signals (Nasonovia ribis-nigri)18,19, whiteflies (Trialeurodes emitted from the damaged willows2. Subsequently, vaporariorum)20 and armyworms (Spodoptera numerous researchers have shown that plants can exigua, Mythimna pseudaletia)14,21 . intercept and respond to herbivore-induced plant Tissue damage is the most obvious consequence of volatiles (HIPVs) in response to attacks by an herbivore attack. It has been shown that herbivorous arthropods. mechanical wounding, clipping or defoliation of some plant species can be sufficient to induce a Volatile signalling as mechanism of interplant communication 2

release of the volatile compounds responsible for herbivores (caterpillars etc.). The mode of transfer of the information between plants8,22–30. An actionreviewed in 38–40 of such molecules is usually experiment performed by Arimura and his connected withthe influx of Ca2+ in plant cells and colleagues however showed a full response of therefore the depolarisation of their plasma receiving lima bean plants only when they were membrane, which can be caused by action of elicitor exposed to volatiles from the spider mite infested receptors41 or, in case of some FACs, due to their conspecific leaves and not when emitting leaves amphiphilic nature, resulting in the formation of were artificially wounded13. The finding that channel-like structures in the membrane. Ca2+ ions mechanical wounding of plants can be insufficient can further take part in signalling through Ca2+ for a complete induction of HIPVs was confirmed sensing proteins (e.g. and - also by Piesik, who compared VOC responses of like proteins) and MAPK cascades (e.g. wound- several cereal plants that were either infested by the induced protein kinase, WIPK, and salicylic acid- cereal leaf beetles or mechanically injured in form of induced protein kinase, SIPK), resulting in the the scrapping injury, simulating the pattern of beetle induction of primarily JA and ethylene signalling tissue damage. Plants exposed to herbivore not only pathways, as well as transcription of specific defence released higher concentrations of volatiles, but also genes. emitted 13 additional specific HIPVs17. On the other Contrary to described elicitors of chewing hand, it has been showed that continuous arthropods, little is known about elicitors derived mechanical damage, induced by a robotic device from sucking, -feeding arthropods such as (MecWorm), resulted in release of HIPVs that aphids and spider mites. Some of their orally- perfectly matched HIPVs released as response to derived molecules might act as elicitors (e.g. caterpillar (Spodopteralittoralis) feeding31. oligogalacturonides) and it has been suggested that What can we therefore conclude about the Ca2+ influx and membrane depolarisation is triggered mechanism behind induction of the plant signalling as result42. Even though the early signalling HIPVs? Mentioned examples fit into the current mechanisms of these herbivores still needs to be knowledge on generalplant-herbivore interactions, clarified, it is well accepted that signalling pathways perception of herbivores and signalling mechanisms trigger primarily the salicylic acid (SA) response that result in the‘induced resistance’, induction of reviewed in34,38. broad spectrum of plant defence systems, including Other molecules involved in the stress response of release of HIPVs. herbivore attack are active nitrogen species (NO) Mechanical wounding of plants results in a and reactive oxygen species(ROS). Although the disruption of plant tissue and damaging of plant specific role of NO in herbivore defence signalling is cells, causing molecules from different not completely clear, it has been shown that its compartments to come in the contact with each production and activity is closely related to the other. According to Heil this can trigger a ‘plant phosphorylation events, Ca2+ influx and activity of 43 2- damaged-self recognition’, resulting in induction of SIPK . ROS (O , H2O2 etc.) burst in herbivore injured jasmonic acid (JA) signalling pathways32. Endogenous plants is thought to be related to Ca2+ ions, FACs and plant signals (also named damage-associated protein kinases activity34. molecular patterns, DAMPs) can therefore act as In short, herbivore induced defence responses are elicitors to induce plant immune response33. Upon activated through a complex, and not yet completely herbivore feeding, plants perceive and respond to understood, cross talk of cell membrane herbivory associated molecular patterns (HAMPs) depolarization, ion flux, mitogen activated protein i.e. herbivore-derived elicitors from oral secretions kinases (MAPKs) activation, as well as induction and of feeding herbivores and possibly HAMPs originated production of reactive oxygen species, nitric oxide from a specific pattern of woundingreviewed in 34. and phytohormones (JA, SA, ethylene), result of Several - conjugates (FACs) which are numerous defence related responses, derived from herbivore saliva, such as volicitin including emission of the HIPVs that can act as (conjugate of 17-hydroxylinolenic acid and L- signalling molecules for neighbouring plants. glutamine)35,36, and also other chemical compounds 35 PATHOGEN INFECTION such ascaeliferins , disulfide-bridged peptides (inceptin) and β-glucosidasereviewed in 37, have been Plants can also perceive volatile signals emitted from shown to act as elicitors derived from chewing neighbours attacked by pathogens. Inoculation of Volatile signalling as mechanism of interplant communication 3

lima bean plants with avirulent strain of the attacks. The same mechanism, as with herbivore biotrophic bacterial pathogen Pseudomonas attacks, is proposed here; plants are able to detect syringae resulted in a defence response in and respond to DAMPs. A question that arises from neighbouring conspecific plants. Furthermore, the this is how are plants able to respond differently to same response was observed when systemic artificial wounding i.e. herbivore or pathogen acquired resistance (SAR) to the pathogen was specific? To fully understand this specificity, more artificially induced in emitting plants with application experiments should be performed, focusing on the of benzothiadiazole44. Shulaevet al. showed that unbiased monitoring of the whole range of tobacco plants Nicotianatabacum attacked by responses to the mechanical wounding. Possible tobacco mosaic virus release VOCs capable of mechanisms behind induced specific responses inducing defence response in their neighbours45. could be based on pattern differences and even Infection of maize leaves with four different temporal properties of mechanical damage. pathogenic filamentous fungi species (Fusarium RESPONSE TO ABIOTIC STRESS FACTORS spp.)resulted in the release of several VOCs acting as plant-plant signalling molecules46. As already seen There are only few studies published that indicate for the herbivore attack, mechanical wounding of that plants can emit and respond to the volatiles, the emitting plant alone can be also insufficient to whose induction is based on abiotic stress factors. invoke a full response in receiving plants upon Being sessile organisms, plants are dependent on pathogen infection.Zeringue showed that cotton limited resources leading to constant competition leaves (Gossypiumhirsutum) exposed to with their neighbouring organisms. Allelopathy, mechanically wounded and Aspergillusjiauus phenomena when plants suppress growth or infected leaves only fully respond to VOCs emitted establishment of their neighbouring plants by from infested plants47. This specific pathogen- emitting specific chemicals into the environment, is induced release of signalling VOCs corresponds with a known mechanism of plant competition52. There mechanisms of plant immunity that are triggered are few studies indicating that plants can also upon pathogen attack. It is well established that respond to non-allelopathic volatile signals from plants able to detect pathogen and microbe- their neighbours, perceiving an increasing lack of associated molecular patterns (PAMPs and MAMPs) space, resources and closeness of other plants and such as fungal chitin, bacterial lipopolysaccharides, consequently responding with a change in growth. peptidoglicans and flagellin with activation of the Ninkovicet al. showed that interplant VOCs mediated surface-localized pattern recognition receptors communication between two barley cultivars (Alva (PRRs) that can be either receptor-like kinases or and Kara) affects biomass allocation in the individual receptor-like proteins reviewed in 48. Such activation plants53. It has also been shown that tobacco plants results in a burst of ROS, Ca2+ influx, activation of can perceive and respond to the ethylene released Ca2+-dependent and mitogen-associated protein by other plants when grown in high dense cultures54, kinases, leading to the induction of SA 55. Numerous abiotic stress factors, such as light, (biotrophicpathogens) or JA and ethylene temperature, UV and readiation are known to (necrotrophic pathogens) signalling pathways, influence the release of VOCs 56. Recently Yao et al. induction of a defence response and potentially to a showed that UV irradiated A. Thaliana plants systematic acquired resistance. This primary immune released volatiles that caused a response in response is called PAMP-triggered immunity (PTI) neighbouring tobacco plants57,58. reviewed in 49, 50.Pathogens are able to suppress PTI with active molecules, called effectors, which can trigger As we can see VOCs detected by neighbours are the second line of the plant immune response mainly produced as part of an immune response or (effector-triggered immunity, ETI). However, since as part of a response to different stress conditions, ETI usually results in basal defence responses, such such as herbivore and pathogen attack, UV radiation as localized programmed cell death51, it probably and increasing lack of resources. It is not possible to does not play an important role in inducing plant- show exactly what triggers the production of plant- plant signalling VOCs. plant signalling VOCs or what elements are involved On the other hand, it has been shown that in their induction and production pathways and how mechanical wounding alone can trigger the release these differ from other regulatory pathways of of interplant VOC signals29 linked to pathogen induced defence. Furthermore, it is very likely (as Volatile signalling as mechanism of interplant communication 4

already mentioned in the introduction) that the signalling and regulation of VOCs and their role in primary function of such VOCs is not neighbour emitter plant defence, a better understanding of signalling, but rather more acknowledged defence specific cues and signalling pathways is necessary. responses (e.g. attraction of carnivorous insects) or within plant signalling. To better understand the

Fig. 2: Cues for release of VOCs responsible for interplant communication, their recognition and signalling pathways leading to the induction of VOCs. Herbivory, pathogen infection and abiotic stress factors have been shown to induce emission of VOCs that play a part in interplant communication. Faded arrows represent pathways that still need to be elucidated or confirmed. Shaded areas include elements involved in cross-talk. Identity of signalling molecules What is the identity of VOCs that were found to reported to be VOC molecules mediating the induce a response in the receiving plants? The most communication between several plant species; commonVOCs involved in the interplant signalling homoterpenes(3E,7E9)-4,8,12-trimethyl-1,3,7,11- are methyl salicylate, oxylipin metabolites methyl tridecatetraene(TMTT) and (3E)-4,8-dimethyl-1,3,7- jasmonate and cis-jasmone as well asterpenoid and nonatriene (DMNT), (E)- β-ocimene green leaf volatile compounds (table 1). and ocimene and (E)-β-farnesene (Table 1). Terpenoidspresent the largest class of secondary metabolites in plants11. They are synthesised via two The second big group of interplant signalling VOCs independent alternative pathways from a five- are (GLVs) which are six-carbon carbon compound isopentenyldiphosphate (IDP) and compounds including the alcohols, aldehydes and its allylic isomer dimethylallyldiphosphate (DMAPP). their esters, formed via hydroperoxidelyase pathway The melvalonic acid (MVA) pathway takes place in of oxylipin metabolism reviewed in 62. GLVs involved in the cytosol, while the 2-C-methyl-D-eryrhritol 4- the plant-plant signalling were shown to be released phosphate (MEP) pathway occurs in the in several plant species in response to herbivore plastidsreviewed in11,37,61. Several were attacks or artificially induced mechanical wounding. Volatile signalling as mechanism of interplant communication 5

The most commonly released compounds are densities8,54, methanol, proposed to be a signal different forms of hexenal, (Z)-3-hexen-1-ol, (Z)-3- transmitter between tobacco plants upon hexen-1-yl acetate and (Z)-3-hex-3-enyl acetate mechanical wounding29, methacrolein, emitted by (Table 1). Other VOCs that often play a part in plant- Artemisia tridentate upon mechanical wounding30, plant signalling are methyl salicylate (MeSA), benzothiazole, an aromatic heterocyclic compound phenolic compound of salicylic acid, and oxylipin shown to act as a signal between populous trees26 metabolites methyl jasmonate (MeJA) and cis- and nonanal, an alkyl aldehyde released fromthe jasmone. Less common are phytohormone ethylene, lima bean plant in response to exposure to P. which was found to be emitted after the defoliation syringae44. of black alder and from tobacco plants grown in high

VOC plant species ecological context ref. (Z)-3-hexenal Chrysanthemum cinerariaefolium Mechanical wounding, laboratory 27,28

(Z)-hexenal Zea mays Mechanical wounding and herbivory 25 (caterpillars), laboratory (E)-2-hexenal Chrysanthemum cinerariaefolium, Mechanical wounding and herbivory 27,28,30,63 Artemisia tridentata(as emitter), (spider mites), field and laboratory Nicotianaattenuata(as receiver), Phaseoluslunatus

(Z)-3-hexen-1-ol Chrysanthemum cinerariaefolium, Mechanical wounding and herbivory 7,16,25,27,28,6 Zea mays, (leafminers, caterpillars, spider mites), 3 Phaseoluslunatus, laboratory Arabidopsis thaliana,

(Z)-3-hexen-1-yl acetate Zea mays, Mechanical wounding and herbivory 7,25,27,28,63,6 green leaf volatiles leaf green Chrysanthemum cinerariaefolium, (caterpillars, spider mites), laboratory 4 Arabidopsis thaliana, Populusdeltoides × nigra, Phaseoluslunatus (Z)-3-hex-3-enyl acetate Phaseoluslunatus Herbivory (beetles), laboratory and field 65

Several GLVs Triticumaestivum, Mechanical wounding and herbivory 17 Avenasativa, (Oelemamelanopus, beetles), laboratory Hordeumvulgare ocimene Phaseoluslunatus (as emitter), Herbivory (leafminers), laboratory 16 Arabidopsis thaliana (as receiver) (E)- β-ocimene Phaseoluslunatus, Herbivory (spider mites), defoliation, 8,13 Alnusglutinosa laboratory and field

(3E)-4,8-dimethyl-1,3,7- Phaseoluslunatus, Herbivory (leafminers, spider mites), 8,13,16 nonatriene (DMNT) Arabidopsis thaliana (as receiver), defoliation, laboratory and field

Alnusglutinosa

terpenes

(3E,7E9)-4,8,12- Phaseoluslunatus, Herbivory (leafminers, spider mites), 8,13,16 trimethyl-1,3,7,11- Arabidopsis thaliana (as receiver), defoliation, laboratory and field tridecatetraene(TMTT) Alnusglutinosa

Volatile signalling as mechanism of interplant communication 6

(E)-β-farnesene Chrysanthemum cinerariaefolium Mechanical wounding, laboratory 27,28

Methyl salicylate (MeSA) Nicotianatabacum, Mechanical wounding, UV radiation, 26,44,45,58 Arabidopsis thaliana, exposure to pathogens (Pseudomonas Phaseoluslunatus, syringae, tobacco mosaic virus), Populussimonii × Populuspyramidalis laboratory and field

Methyl jasmonate Artemisia tridentate (as emitter), Mechanical wounding, UV radiation, 23,26,30,58 (MeJA) Lycopersicomesculentum(as receiver), laboratory and field Nicotianaattenuata(as receiver), Arabidopsis thaliana, Populussimonii × Populuspyramidalis

Cis-jasmone Zea mays, Mechanical wounding and herbivory 66,19,25 Ribesnigrum(as emitter), (caterpillars, alphids), laboratory Viciafaba

Ethylene Alnusglutinosa, Defoliation, plants growing in high 8,54

other Nicotianatabacum densities, field and laboratory

Methanol Nicotianabenthamiana Mechanical wounding, laboratory 29

Methacrolein Artemisia tridentata(as emitter), Mechanical wounding, laboratory and 30 Nicotianaattenuata(as receiver) field

Benzothiazole Populussimonii × Populuspyramidalis Mechanical wounding, laboratory 26

Nonanal Phaseoluslunatus Exposure to pathogen (Pseudomonas 44 syringae), laboratory and field

Table 1: Identity of VOCs involved in interplant communication.VOCs are listed with plant species, known to emit and receive them, and ecological conditions, under which their release wasinduced.

Volatile signalling as mechanism of interplant communication 7

Release of VOCs trigger the same response under field conditions, where not only the concentration would probably be When plants suffer herbivore or pathogen attacks, lower, but also natural occurrences such as or are under the abiotic stress conditions, such as radiation, presence of air pollutants, wind and strong UV radiation, a mechanical wounding alone temperature changes could contribute to a lesser can already enable some volatile compounds to be amount of volatile molecules reaching the receiving released from the disrupted cellular compartments, plant. Indeed, air pollution has been found to where they were stored. Such VOCs are released influence the transfer of signalling molecules; from the damaged sites immediately. Such VOCs can Blandeet al. showed that ozone concentrations be also emitted from the undamaged sites of plants commonly encountered in nature (80 ppb) with some time delay regarding the attack67. VOCs significantly decreased the distance over which VOCs can also be released as de novo synthesised could be transmitted between lima bean plants. This compounds. This usually occurs as part of the was explained by ozone dependant degradation of systemic defence response. Such VOCs can also be 69 HIPVs in the atmosphere . On the other hand, emitted with some time delay from the undamaged 67 ozone was also shown to cause increased emission parts of the plant. . They are released through leaf 70 of VOCs .Some VOCs can be oxidised or otherwise stomata and, in case of the lipophilic volatiles, such processed in the atmosphere during transport. GLVs as terpenes, also through the membrane of are oxidised by ozone, NO and OH and are epidermal tissues and from other structures, such as 3 68 estimated to remain intact for only a few hours after trichomes . Most of volatile compounds that take 37 release . Whether such modified VOCs have part in interplant communication are synthesised de ecological function is unknown. novo or from the already stored precursors under control of the signalling pathways triggered by the When considering VOCs another factor that has to previously described cues. be taken into account is the properties of volatile GLVs for example are emitted from the disrupted molecules themselves. Small molecules, such as leaf and stem tissues very rapidly (within a few ethylene, are easily diluted in the atmosphere as reviewed in 37 seconds to few minutes ) after an attack. they diffuse very rapidly. Instead of long distance Such a rapid response is thought to result from the signalling over open space, ethylene, a singular contact of substrate and enzymes that are under signalling molecule, is more successful in signalling normal conditions stored in separate compartments within enclosed spaces, such as under canopies54. yet encounter each other only after tissue damage. GLVs, terpenes, MeJA and MeSA on the other hand Regarding the substrate (free fatty acids) for the are heavier compounds with less volatility71 and formation of GLVs, it has been suggested that they consequently they do not diffuse in every direction are formed upon tissue damage via hydrolysis of but rather stay closer to the ground meaning that galactolipids. Decompartmentalization however is the release of lower concentrations of such volatiles not always necessary for the formation of GLVs; could possibly still reach the receiving plants. GLVs were observed to be released also from the undamaged plant tissues as part of what is thought After initial laboratory oriented experiments, several to be a systemic response of plants under herbivore studies were performed in the field, under natural attack reviewed in 37,62. on the other hand are conditions, conforming the transfer of signal for normally released with a several hour delay after several VOCs and plant species (table 1).Karbanet al. attack, after induction of biosynthetic genes such as observed how neighbours of clipped sagebrushes synthase (TPS), resulting in complete de became herbivore resistant; they showed that a novo synthesis of the volatile compounds37. signal could be transferred as far as 60 cm from the emitting plant. Furthermore, they observed that In order to convey a message to the neighbouring most of the emitting sagebrushes had conspecific plants, released VOCs must be successfully neighbours closer than that72. Heilet al. performed transported to the receivers. Many of the initial experiments on lima bean plants and observed experiments on interplant communication were transmission of airborne resistance up to 50 cm from performed under laboratory conditions, usually the emitting plant, after which it dropped drastically, utilising concentrations of VOCs far above naturally and at a distance of 100 cm no significant increase in occurring ones. As a result it was questioned resistance of plants was observed4. Transmission of whether aerial transport of tested compounds would Volatile signalling as mechanism of interplant communication 8

the signal for over 3 m from the emitting plant was active epimer was observed after clipping a observed between maize plants as well as between sagebrush plant in comparison to the control some other grasses, such as wheat, barley and bushes6 and the trans to cisepimeric switch oats46. appeared to elict defence responses in tobacco plants more effectively than either epimer alone74. Research in the last couple of years has shed new Such specific patterns of VOCs of different light on just how complex interplant signalling is. concentrations offer much wider variety of different That plants can send specific messages and that possible signals and could be the answer to the receiving plants respond differently to different coding and perception of numerous specific signals was mentioned already in the chapter about messages. cues recognition. Plants can therefore send a The concentration of volatile compounds seems to message that will vary depending on the type or play an important role in plant-plant signalling. even species of the attacker. Furthermore, it has Perceived concentration of VOCs not only depends recently been shown that plants can respond on the emitted concentration at a specific moment differently depending on the (genetic) relatedness of 9 but also on accumulatively low signal concentrations the emitting plant . This was demonstrated by a over a long exposure time. Shiojiri showed that study performed using three annual plant species exposure to trace amounts (less than 140 pptV) of (Lipinus nanus, Sanpis arvensis and Achyrachaena GLVs over 3 weeks induced defensive response in mollis) where defence responses of receiving plants undamaged A. thaliana plant7. An experiment where growing next to damaged neighbours depended on 5 lima bean plants were exposed to different genetic relatedness . concentrations of MeSA over 6 and 24 hours showed How are plants able to emit and differentiate that an exposure to very low concentrations for between all this different information utilising only a longer exposure time resulted in a strong defence few so far described volatile compounds? It has response, which was not observed when plants were become evident that signalling VOCs are usually exposed to the same concentration of VOC for a released as specific blends of different compounds, shorter period75.These experiments indicate that a often exhibiting specific concentrations of each of reviewed in 28 plant-plant signalling can involve an accumulation of them . Chrysantenum cinerariaefolium the VOCs in the receiving plant. It is evident that the seedlings exposed to volatiles from damaged exposure time can be critical for a signal transfer, conspecific neighbours exhibited exactly the same possibly enabling detection of signals with very low defence response (i.e. induction of transcription of concentration over time. This could mean that the 13-LOX, DXS, CPP and AOD genes as well as actual distance for a successful transfer of a signal enhanced pyrethrin content) as seedlings exposed to could be greater than shown in experiments so far, the blend of volatiles matching concentrations and provided VOCs are released for long enough time. compounds of naturally released ones. However Furthermore it opens a question whether some when even one of the five VOCs was removed from VOCs involved in the interplant signalling have been the blend, gene expression was drastically reduced. overlooked because of their low emission Furthermore, the blend of volatiles was only fully concentration. The bottom line is that such findings effective at specific concentrations; an increase or provide a good reference for the importance of the decrease of concentrations of VOCs resulted in 27 long-term experiments in the future studies. reduced gene expression . Ruther et al. showed the other possible role of volatile blends by RECEIVING AND RESPONDING TO THE demonstrating that ethylene can act as a synergist to MESSAGE GLVs, inducing 2.5 fold increased defence response in maize plants when added to (Z)-3-hexenol73. DIRECT RESPONSE TO THE VOLATILE SIGNALS Different forms, i.e. epimers of the released volatiles, could also have role in different signal The idea of interplant communication was first transmission. Cisepimer of MeJA, cis-MeJA, is triggered by the observations of the response biologically more reactive and therefore actions carried out by the neighbours of attacked thermodynamically less stable, normally resulting in plants. Rhoades observed herbivore resistance of rapid epimerization to the more stable trans willow trees growing in close proximity of trees configuration. Approximately a 10 fold increase of infested with tent caterpillars2 and Baldwin and Volatile signalling as mechanism of interplant communication 9

Schultz reported increased concentrations of response, such as the attraction of herbivore phenolic compounds in undamaged poplar ramets predators and parasitoid insects. It has been shown and sugar maple seedlings enclosed together with that cotton seedlings exhibit increased attraction to damaged conspecific plants1. Numerous reports predatory mites when exposed to volatiles from followed describing the induction of plant defence seedlings, infested with herbivore mites78. Increased responses that were either exposed to volatiles attraction to predatory and parasitoid insects (i.e. emitting from neighbouring plants of the same or ants and wasps) was also shown for lime bean plants different species, or to the applied compounds exposed to the VOCs emitted from herbivore- corresponding the identity of VOCs, reported to be damaged lima beans65. Birkett observed that the involved in interplant signalling. application of cis-jasmone to intact lima bean plants induced production of several VOCs, including (E)-β- A common response of receiving plants is acquired ocimene. As a consequence plants became more resistance, shown as a reduced damage by the attracted to aphid parasitoids66. Interestingly, newly attackers. Dolchet al. showed under field conditions emitted VOCs from the receiving plant can also that defoliation of alder trees resulted in a reduced attract pest insects; in a study performed on maize amount of damage by alder leaf beetle and a plants, exposed to volatiles from fungus infected reduced number of beetle eggs per leaf in the 24 cospecific neighbours, Piesik showed induced neighbouring trees . Air transfer experiments emission of several GLVs and terpenes and between clipped and undamaged sagebrush plants consequently increased attraction to insect pests O. recently showed that unclipped plants treated with melanopus46. air from clipped ones exhibitedreduced damage in response to naturally occurring herbivory76. Volatiles Composition of VOCs, emitted from the receiving emitted from whitefly infested tomato plants plants,is often similar to the volatiles that were induced a bacterial resistance in neighbouring shown to take part in the interplant signalling. This is plants, showing cross-kingdom effect of the not unusual, since it is very likely that the same interplant signalling20. Received VOCs were also volatiles that are emitted as part of the defence shown to make plants more resistant to fungal response, take part in several other modes of action disease77, viral45 and bacterial29 infections. Several (Fig. 1), such as within plant signalling and attraction compounds, that are part of the induced direct of predatory insects. However one question that defence response in receiving plants, were shown to remains to be answered is whether these volatiles accumulate or exhibit increased activity after can also be perceived by the neighbouring plants exposure to interplant signals. Tomato plants and induce a response. Such a chain of action would exposed to MeJA, emitted from sagebrush plants, mean that the message from the original emitter responded by increased production of the plant would be able to reach neighbouring plants proteinase inhibitors23. Black alders, shown to over a much larger area. acquire resistance to herbivory when exposed to Another question that we can ask concerns the defoliated conspecific trees, also showed an increase mechanism by which VOCs are perceived. The in the activity of proteinase inhibitors and catalase similarity in the compositions of re-emitted and when exposed to MeJA. Incubation with ethylene received VOCs from attacked plants suggest that however resulted in production of phenolics, as well response mechanisms of receiving plants are as proteinase inhibitors8. Wild tobacco neighbours of passive, simple re-emission of the received volatiles. clipped sagebrushes were shown to have increased Such passive adsorption and re-emission was levels of polyphenol oxidase, a defensive oxidative observed in the case of birch trees (Betula spp.), enzyme, in case of a seasonalt attack by cutworms which were shown to adsorb and re-emit arthropod- and grasshoppers6. Hu observed that conspecific repelling terpenes, emitted by neighbouring neighbours of Populussimoniix P. pyramidalis Rhododendron tomentosum79. Chohet al. showed cuttings showed increased levels of similar results for conspecific plants; when lima bean phenolicspyrochatechol, chloragenic acid, gallic acid plants were exposed to the volatiles of plants and p-hydroxyl benzoic acid26. infested with spider mites, the emission of a blend of Plants can respond to the received signals also by volatiles, similar to the one released from infested emitting volatile compounds themselves; as already plants, was observed. Receiving plants were shown mentioned, this can result in an indirect defence to emit volatile compounds even when their Volatile signalling as mechanism of interplant communication 10

production was inhibited with a protein-synthesis lipoxygenase 2 (LOX2)77. An increase in LOX product inhibitor. From this, the authors deduced that plants (Z)-3-hexenyl acetate was shown to result in the passively emitted volatiles that had previously been conversion of exogenous GLV (Z)-3-hexenol, adsorbed. However leaves that were previously indicating that VOCs not only trigger response, but exposed to volatiles from infested leaves emitted can also be further processed81. Furthermore, JA- larger amounts of volatiles and were also more dependent and independent pathways were attracted to predatory mites, which indicates that suggested to play part in GLVs induced response77. both the active and passive modes of action play a MeJA was shown to be responsible for upregulated part in volatile release by receiving plants15. transcription of MPI gene, i.e. gene expressing maize proteinase inhibitor (protein responsible for It is well accepted that plants also actively respond inhibition of digestive proteases of insects in maize to perceived signals; this is evident from the plants), IGL gene (coding enzyme responsible for accumulation of defensive compounds in the production of indole), FPSgene (coding enzyme receivers. Furthermore, several transcriptional responsible for synthesis of sesquiterpenes)81, AOS analyses and monitoring of expression profiles and HPL, coding enzymes in phytooxylipin pathway identified numerous genes that show induced and VSP1 genethat plays a part in ethylene and JA expression upon preception of volatile signals. signalling pathways60. By conversion into jasmonic Activation of five defence genes, i.e. genes for the acid and jasmonoyl isoleucine exogenous MeJA can pathogen-related (PR) proteins PR-2 and PR-3, also trigger signal transduction, leading to the lipoxygenase (LOX), phenylalanine ammonia-lyase activation ofthe defensive systems, e.g. VOCs (PAL) and farnesyl pyrophosphate synthetase (FPS), emission in receiving plants82. were detected in a study by Arimura et al., who Allo-ocimene was shown to induce expression of exposed lima beans to volatiles from conspecific 13 genes for chalcone synthase (CHS), affeic acid-O- leaves infested with spider mites . The same genes methyltransferase (COMT), diacyglycerol kinase 1 were also expressed in lima beans when exposed to (DGK1), glutathione-S-transferase 1 (GST1), (E)-2-hexenal. Exposure to GLVs (Z)-3-hexenol and lipoxygenase 2 (LOX2)77, genes AOS and HPL, coding (Z)-3-hexenyl actetate resulted in different patterns for enzymes playing part in phytooxylipin pathway, of gene expression – (Z)-3-hexenol activated genes and PR-3 and VSP1 that play a part in ethylene and PR-2, LOX, PAL and FPS and (Z)-3-hexenyl actetate 60 63 jasmonic acid signalling pathways . genes LOX, PR-3 and FPS . Interestingly, only the Exposure to MeSA induced upregulated expression transcript of PR-2 gene was detected in receiving of PR-1 gene, molecular marker for acquired leavesexposed to VOCs emitted from mechanically 45 13 resistance , as well as PR-1 and PR-2 genes, markers damaged leaves .This indicates that a different of salicylicacidsignalling60. MeSA itself can play a part combinations of genes are triggered in the receiving in such signalling, as it has been shown that it can be plant depending on the signals and confirms that converted back to salicylic acid45. plants can not only emit cue specific signals, but also Cis-jasmone triggered transcription profiles with perceive them as different and act accordingly. upregulatedgenes for a cytochrome P450 and Experiments using plants whose GLVs production CYP81D1183, and several genes that were shown to was genetically silenced further showed that be under control of the transcription factors TGA2, specific gene expression patterns can also be a TGA5 and TGA6, as well as regulatory protein consequence of the suppressive effect of some SCARECROW-like 14 (SCL14), which could be a volatiles; comparison of gene transcriptional consequence of a specific regulatory pathway patterns of tobacco plants after exposure to GLVs- triggered by its perception84. deficient and GLVs-complemented (resembling WT Expression profiles of A. thaliana plants stimulated VOCs blend) volatile blend showed that numerous 80 with methanol showed methanol dependent genes were under the negative regulation of GLVs . upregulation of 484 transcripts, which varied Among others, GLVs were also shown to induce depending on the time during which samples were expression of PAL gene, which plays part in MeSA 81 exposed to methanol, indicating activation of synthesis, resulting in emission of MeSA volatile , multiple signalling pathways at specific times. In genes for chalcone synthase (CHS), affeic acid-O- general, most of the expression activity was methyltransferase (COMT), diacyglycerol kinase detected in the areas of metabolism, cell 1(DGK1), glutathione-S-transferase 1 (GST1) and communication, signal transduction processes and Volatile signalling as mechanism of interplant communication 11

defence genes85. It has been recently shown that induced responses. What is still unknown is the role genes β-1,3-glucanase (BG), a previously unidentified of sensory proteins, most likely membrane- gene (MIG-21), and non-cell-autonomous pathway associated, able to detect VOCs and trigger further protein (NCAPP) might be of specific importance, as response. So far, the only known receptor able to they are especially affected29. detect volatile compounds in plants is a receptor for Zhang et al. have recently published a full gaseous ethylene38. Further research focusing on the transcriptional analysis of A. thaliana in response to early signalling of receiving plants is therefore the leaf miner-induced volatiles from a lima bean needed for a better understanding of the molecular plant as well as to the individual volatile compounds signal transduction. that were shown to be emitted from lima bean plants, presenting genome wide response to the PRIMING interplantsignalling. The study showed that Plants sometimes receive and perceive a signal, but transcriptional responses are positively correlated develop a full defence response only after they are with treatment duration; only a few genes were challenged by herbivores or pathogens. Signal enriched after a 24 hour treatment, all related to therefore primes their defences for attacks yet to thedefense responses. However, after 48 hours of occur. Such response could be favourable for plant treatment upregulation of numerous genes fitness, since preparing a defence response is quite associated with both defense and metabolism energetically costly and its direct induction after the pathways occurred. Furthermore, they have shown signal perception can mean energetic waste for that ethylene and JA pathways are involved in plants if they are not challenged soon after. It looks detecting VOCs and that the ethylene pathway in the like plants respond to the volatile signals in complex 16 receiver plants is essential for communication . ways, often exhibiting a mix of priming and directly Gene transcription and expression analyses are able induced defence responses. to provide some insight into which signalling Engelberth et al. were the first to clearly pathways and which phytohormones are involved in demonstrate the primingphenomena for airborne the downstream detection of VOCs and in forming signalling; they showed that corn seedlings, the defence response. Much less is however known previously exposed to GLVs, produced more about upstream signalling, perception of VOCs and terpenoids and JA in comparison with non-exposed the transduction mechanisms. Some volatiles, such seedlings when mechanically damaged or induced 25 as MeJA and MeSA, were shown to be converted to with caterpillar regurgitant . Heil et al. showed the JA or SA and therefore probably have the role of similar results for the production of extrafloral signalling molecules/phytohormones themselves. It nectar in lima bean plants; leaves that were is very likely that the activation of defence genes by previously exposed to volatiles from beetle-damaged VOCs can be also mediated via signalling processes conspecific shoots, showed a significantly higher such as protein phosphorylation/dephosphorylation, production of extrafloral nectar when they were Ca2+ influx and burst of reactive oxygen species. mechanically damaged in comparison to non- 10 There is some evidence that supports this exposed ones . Another example of priming as assumption. Arimura et al. showed that the response to interplant airborne signalling was expression of monitored VOCs-dependent defence observed in a natural population of lima bean plants; genes required Ca2+ influx and protein when challenged with pathogen infection, plants phosphorylation13. Furthermore Asai et al. previously exposed to the volatiles from neighbours demonstrated an increase of cytoplasmic free Ca2+ with induced systemic acquired resistance showed concentration in leaves of A. thaliana exposed to the significantly stronger expression of the pathogen- volatiles. When plants were exposed to acyclic related protein 2 (PR-2) than non-exposed plants, 44 volatile compounds (β-ocimene, β-myrcene, DMNT), and consequently exhibited better resistance . such a rise in concentration was shown to result in Priming was also observed in woody plants; hybrid Ca2+release from the intracellular compartments. In poplars showed priming of JA, proteinase inhibitor 64 the case of (E)-2-hexenal however, Ca2+ influx was and terpene volatiles synthesis . mediated via ROS production by natural oxidation86, Analyses of gene expression are consistent with indicating that at least two different signal before mentioned phenomena. Even when changes transduction processes are involved in forming VOC in the phenotype are not immediately observed, 12

transcription patterns of the defence related genes stronger and earlier induction upon subsequent indicate that signals are perceived by the receiver attack87. plants. Microarrays enriched in herbivore-regulated The mechanisms behind the airborne-induced genes of the native tobacco plants, neighbouring priming are still mostly unknown88. Jaskiewiczet al. clipped sagebrushes, showed transcriptional showed that chromatin modification might play a responses, however presence of the defensive role in the in-direct activation of genes. Treatment chemicals or proteins in tobacco plants was not with an analogue of SA or pathogen infection detected.However when the same plants were induced chromatin modifications on promoters that challenged by application of caterpillars, accelerated are usually connected with activity of genes; production of trypsin protease inhibitors was however observed defence-related genes remained observed30. Similar non-direct activation of the inactive. A strong correlation between histone genes was observed in maize plants, exposed to modification patterns and gene priming was VOCs from caterpillar infested neighbours; 10 observed, indicating that histone memory could play defence-related genes were identified, showing a part in gene modifications relating to priming89.

Fig. 3: Schematic overview of reported response actions of receiving plants to VOCs and indication of signalling mechanisms involved in VOCs perception further processing of signal. Received volatile signals can be either passively adsorbed and reemitted, or actively processed resulting in induction of defence response. This can be in form of direct induction of defence actions, such as emission of defence-related volatiles and accumulation of attacker inhibiting compounds, or in form of priming, phenomena when defence gene expression is fully activated only after plants are subsequently challenged. Green shaded area represents inside space of plant tissue.

Mechanisms of underground interplant communication 13

Mechanisms of underground neighbour; when grown alone, plants released a higher number of defence-related proteins, whereas interplant communication plants growing with neighbours exhibited a A significant part of the plant’s structure lies decreased repertoire of these proteins and an underground. It is well known that this is where increased secretion of specific defence-related numerous interactions with other organisms, such as proteins. Stress related proteins showed the reverse symbiotic microorganisms and fungi take place. It trend; both, the number and acumulative secretion has also been shown lately that plants can actively of peroxidises decreased when plants were grown shape and control such interactions90. Despite that, with neighbours. Furthermore, specific proteins were found to be only secreted when A. dumosa was there is still little known about the interplant 95 interactions and possible communication below neighbouring a specific neighbour . These findings ground. Evidence for interplant communication has indicate that plants are able to biochemically sense been so far found for the signalling through roots and distinguish similar or different neighbours. and common mycorrhizalnetworks; however most of Ciszaket al. recently proposed that plant recognition the mechanisms behind them so far remain abilities can result in the establishment of social unknown. behaviour; swarming behaviour was observed in alignment patterns of the roots of neighbouring Signalling through roots maize plants. Root apices were observed to act as decision-making centres, establishing correlations in It has been shown that roots respond to growth patterns96. More data should however be neighbouring roots in a specific and actively obtained to confirm that root based interplant controlled manner reviewed in 91. Most studies on root recognition can indeed lead to such social behaviour based interaction between plants have focused on and if so, elucidate how this process is mediated. competition-based behaviour. Such interactions can Evidence exists that plants can also emit defence- depend on the species and the identity of the related cues through roots that can be perceived neighbours92. Different root-root behaviour was and answered by neighbouring plants. Dickeet al. shown for different desert shrubs; roots of showed transfer of the herbivore-induced signal Ambrosia dumosa detect and avoid roots of between lima bean plants. Lima bean plants infested conspecific plants, whereas Larrea tridentata roots with spider mites were placed in distilled water with exhibit inhibition of neighbouring Larrea and their roots. Subsequently, roots of uninfested Ambrosia roots93. Furthermore, there is an conspecific plants were placed in the same water, accumulating amount of the evidence that roots can which resulted in their increased attraction to the recognise kin and relatedness of the neighbouring predatory mites, indicating that above-ground plants and respond accordingly. herbivory can lead to the root changes and that Cakileedentula showed increased allocation to roots chemical information can be transferred through when groups of non-related plants were grown 97 soil . Even though this study is a good indication together. Such phenomena was not observed in case that root transfer of signals through soil is a likely of the siblings94, indicating that plants can possibility, it was performed under laboratory discriminate kin in competitive interactions and that conditions. Plants were left in the water with their roots can very likely provide cues for such kin roots for seven days, which could have caused a recognition.Root allocation is usually connected with physiological stress and consequently responses the increase of below-ground competitive ability; such as changed root permeability.98 therefore these resultsare consistent with kin Similar observations were reported by Guerrieri et selection. To better understand root recognition of al., who observed that healthy broad bean plants plant neighbours at the molecular and biochemical (Viciafaba) grown in the same pot as conspecific level, Badriet al. recently analysed root secreted plants infested with the aphid exhibited metabolites and proteins involved in early plant increased attraction to the parasitoid Aphidius ervi. neighbour recognition, using A. thaliana co-cultured Such a phenomenon was not observed when root with different ecotype of A. thaliana or Capsella contact was prevented among plants. Furthermore, rubella neighbours. They observed a significant a defence response of intact plants was triggered difference in secretion profiles of plants growing when they were placed for 24 hours in hydroponic alone in comparison with plants growing next to a Mechanisms of underground interplant communication 14

solution in which infested conspecific plants were from the neighbouring plants exposed to osmotic grown previously. Such response was not observed stress, and are also able to induce responses in in the case of hydroponic solution previously used to additional unstressed plants. Plants might therefore grow intact plants, indicating that water soluble be able to increase their readiness to future drought exudates from roots of infested plants are situations or osmotic stress. Furthermore, responsible for the signal transfer and triggering of neighbouring plants were shown to acclimate to the induced defence response in intact plants99. In stress cues and re-open their stomata after a few comparison to the research performed by Dickeet hours, showing that such induced readiness is al., time of exposure of uninfested plants to medium contemporary in absence of actual stress. This could in which infested plants were grown was much be of relevance to fitness since open stomata plays shorter (24 hours versus 7 days97,99), which could an important role in gaseous exchange, which is an meanthat different signalling pathways were taking essential part of plant energy metabolism100. Even part in signal transduction and recognition. though it is indeed very likely that the signal transfer Molecular mechanisms as well as identity of was conducted through roots of plants, which were compounds, responsible for such communication, made to connect in a very specific manner, air space remain unknown. Furthermore, even though part of between plants was not limited. Stomatas of the mentioned experiments were executed using soil emitting induced plants were closed, which is also in as medium, most of the discussed evidence for favour of the root-root communication; however transport of defence cues remains based on the volatiles could be emitted and absorbed through experiments using aqueous media and plant tissue. Signal transport via air therefore cannot hydroponics.This might not accurately portray actual be completely ruled out in this case of the interplant behaviour of plants; such artificial medium might signalling. affect root structure and plant response to As we can see, there is strong evidence supporting environmental cues, since they present a different the communication of plants through their roots. On environment, lacking space and aggregates present the other hand, there is also data lacking on the in soil and not taking into account microbal activity mechanisms of molecular signalling pathways or fluctuations in temperature, CO , ion 2 behind the signal transfer and on the identity of the concentration and water. signalling compounds. Herbivore attack is not the only topic of interplant Patterns of volatile response to herbivore attack are root-root signalling. Recently Falik et al. showed that in some ways similar for both above and below plants can also emit and perceive stress related ground. Below ground volatile emissions were signals. In their experimental set-up, individual split- shown to be able to protect plants by attracting root pea plants (Pisium sativa) were planted in two entomopathogenic nematodes, much as above pots; separate roots were planted in an induction ground VOCs can do with attracting parasitoids and pot, which was subjected to osmotic stress and predatory insects. One such compound, either in a pot with five or one target plant, sharing (E)-β-caryophyllene, was identified as their rooting volume. In case of the one target plant, an insect-induced below ground plant signal, also split-rooted, one of the roots of target plant was emitted by maize roots in response to the feeding by further connected with a new target plant, enabling beetle larvae. It exhibited high ability of the diffusion monitoring of the root signal transfer from induced (up to 10 cm) in sand-containing moist soil, which, plant through receiving plant to its neighbours. As together with its chemical stability, seem to make it targeting pea plants, bermuda grass Cynodond exceptionally suitable as a below ground signal101. actylon, hairy crabgrass Digitaria sanguinalis and Another below ground volatile, pregeijerene (1,5- buffalo grass Stenotaphrum secundatum were used. dimethylcyclodeca-1,5,7-triene), was observed to be The response of the induced and neighbouring emitted from the citrus roots upon larval Diaprepes plants to the osmotic stress was monitored by abbreviatus feeding and also shown to attract recording the stomatal aperture kinetics. All of the entomopathogenic nematodes102. target plants exhibited stomatal closure when they Given good diffusion rates and considering the were neighboured with stressed plants, which examples from volatile above ground signalling, it indicates that unstressed plants are able to perceive may be that mentioned volatiles play a part in the and respond to the stress-induced cues emitted interplant root to root communication; that they can Mechanisms of underground interplant communication 15

be perceived by roots of neighbouring plants and with metals, enabling slower decrease in their consequently induce their defence response. This is concentration over increased distance107. of course purely speculative; however it offers Several published studies support this model. So far interesting opportunity for possible further studies. studies have focused on the networks formed by Furthermore, even if these specific volatiles play a arbuscularmycorrhizal fungi, i.e. fungi known to role only in attracting parasitoidic nematodes, they associate with more than 80% of the vascular plants still give clues to the type of molecule that may be and have low host specificity, commonly resulting in worth looking into as possible candidates of root to communities consisting of several species of root signalling. plants103. Experiments comparing accumulation of several hydrophilic and lipophilic allelopathic Signalling through underground compounds after their diffusion either through the common mycorrhizal network (CMN) soil or through the CMNs showed that accumulation of compounds was greater when plants were Mycorrhizal fungi are commonly symbiotic, growing in the soil with CMNs. Furthermore, CMNs dependent on carbon uptake supplied by plants and significantly increased the distance through which providing nutrients, such as nitrogen and compounds were able to commute; a gap of 1.5 cm 103 phosphorus to hosts . Numerous plants can be was enough to prevent transfer of observed attached to the same network, creating opportunity compounds through soil, whereas transfer through for some plants to ‘cheat’; contribute to less or none CMNs was observed in all tested distances between of the carbon to the network, however still uptake plants, up to a maximum of 12 cm108. This indicates supplied nutrients. Interactions in form of the that CMNs might be especially important for sanction mechanisms were reported to exist transferring compounds of low water solubility in between plants and between plants and fungi in systems where roots of plants are far apart or are 104 such cases . It has been recently shown that plants not in direct contact. can also exhibit kin recognition in the mycorrhizal Interplant communication through CMNs has also networks; mycorrhizal network size and root been reported to be involved in the transfer of colonization were greater when sibling plants were defence signals. Song et al. observed tomato plants connected in comparison to the non-related ones, (Lycipersicon esculentum Mill.) connected with a 105 resulting in the higher uptake of nutrients . Such mycorrhizal network formed by the fungus Glomus kin selection could be an alternative to sanctions in mosseae. Direct root contact and above-ground air prevention of the cheating, as investment of carbon transfer was prevented between plants. When one in plants closely related could mean increase of the of the plants, connected with a CMN, was infected indirect fitness of plants. Common mycorrhizal with the pathogen Alternaria solani, neighbouring networks (CMNs) also play part in the transfer of healthy plants exhibited increased disease resistance water and nutrients between plants. Such transfer of and activity of defense-related enzymes peroxidise, carbon between roots of ectomycorrhizal tree polyphenol oxidase, chitinase, β-1,3-glucanase, species Betula papyrifera and Pseudotsuga menziesii phenylalanine ammonia-lyase and lipoxygenase. can be bidirectional and possibly regulated by a Furthermore, receiving plants showed a significant 106 source-sink relationship . increase in the expression of six defence genes: It is increasingly acknowledged that CMNs can also genes encoding tpathogen-related proteins PR1, PR- enhance transport of allelopathic and signalling 2 and PR-3, phenylalanine ammonia-lyase (PAL), LOX compounds between plants; a Network Enhanced and AOC. PAL, LOX and AOC play a part in SA and JA Bioactive Zone model proposed by Bartoet al. signalling pathways, indicating their role in the suggests that CMNs act as superhighways, directly transduction of signals leading to an induced connecting plants below ground and therefore resistance in receiving plants109. A study recently increasing the bioactive zones of the infochemicals. published by Babikova et al. used a very similar In comparison to the classical below ground experimental approach to determine whether CMN signalling, such transfer of information could protect can also affect transport of the signal, induced by transported molecules from exposure to the soil herbivore infestation. Bean plants (V. faba) sharing a environment and therefore prevent their fast mycorrhizal connection with conspecific plants degradation, sorption or formation of complexes infested with pea aphids (Acyrthosiphon pisum), showed an increased attraction for the parasitoid Plants as whole organisms – integrated view on the interplant communication 16

wasp (A. ervi); a phenomena that was absent when Comparison of the published data shows that similar plants lacked CMN connections110. plants sometimes exhibited different types of Interplant communication with signal transport interplant communication in different experiments. through CMN presents an attractive mechanism of Lima bean plants infested with spider mites are very plant-plant signalling, however more studies are commonly used as a model system for interplant required to confirm current findings. The identity of communication. In this system, aerial transport of signalling compounds, mechanism of their release, defence-inducting VOCs between neighbouring perception as well as the molecular signalling plants has been shown independently several pathways involved in the induction of defence times13–15,65, always using only isolated above- responses, still need to be discovered. There is ground growing plant parts in the experimental set- already some indication that the transport of up. The exact same system of lima bean plants signalling molecules could be cytoplasmic, following infested with spider mites was also used in an active uptake by hyphae or passive across the aforementioned experiment performed by Dicke et fungal cell membranes. Alternatively, transport can al, where signals were transmitted between plant be apoplastic, if the compounds diffused through the roots in aqueous medium97. Furthermore, attraction fungal cell wall, or surficial, if the compounds of predators, which was the response of diffused through the layer of water on the surface of neighbouring plants in the case of root to root the hyphae. Mycorrhizal hyphae can create channels signalling, was also described in three of the when intertwined together; such cords can be filled experiments with VOC mediated with water, which could also create a transport communication13,15,65. Considering that cues for route for the signalling compounds107. Whether any signalling and responses of receiving plants were the of these suggested mechanisms play a role in the same in all cases, it is quite likely that signalling takes signalling through CMNs is however yet to be place above and belowground simultaneously. determined. Another plant, used in separate experiments describing different ways of interplant communication, is the broad bean (V. faba), Plants as whole organisms – reported to take part in root to root99, as well as 110 integrated view on the interplant mycorrhizal mediated , signalling in response to pea aphid infestations, which in both cases resulted communication in an increased attraction of the parasitoid wasp (A. ervi) to the receiving plant. In the root experiment, As it has been discussed throught this thesis, several carried out by Guerrieri et al. group, the role of types of interplant signal communications exist. aerial transfer on interplant signalling was excluded However, most of experimentatal research focuses by the experiment, where plants were placed in on only a single type of signal transport at a time and close proximity and the contact of the roots was eyperiments have been designed in such a way that prevented by placing plants in separate pots. other types of signal transfer, except the tested one, Resulting responses of receiving plants were similar are prevented. It has rarely been considered that to the responses of the control plants99. In an other possible mechanisms of communication experiment by Babikova et al. on CMNs, aerial probably occur6. On the whole, it seems that transfer was excluded by placing the receiver plants experimental research on certain mechanisms of in polyethyleneterephthalate (PET) bags110. interplant communication has been overlooked. This Interestingly, the experiment by Babikova et al. also appears to be especially true for the transport of excluded any significant influence of root to root signals through CMNs; in laboratory greenhouse signalling110, which is inconsistent with Guerrieri et experiments, sterile soil is very commonly used, al.’s root experiment, who used the same plant- which means that mycorrhizal fungi cannot develop. herbivore system, resulting in the same response of Furthermore, a development of CMNs between receiving plants. Since Guerrieri et al. used sterile plants takes time, so even when natural soil is used, soil in the experiments99, it is not very likely that root experiments performed are usually too short for to root signalling was actually mediated by connections to form. mycorrhizal connection. Considering all this, it is likely that both CMN and root to root interplant signalling can take place; however vary depending Plants as whole organisms – integrated view on the interplant communication 17

on specific circumstances, such as the amount of signals from different parts as the site where signal- time that has passed from induction of the signal. inducing cue originated can be seen in most Separate experiments showed that tomato plants reported cases of underground can communicate via mycorrhizal109, as well as VOC communication97,99,109,110. There is also evidence for mediated aerial transport20. In these experiments, the reverse situation; infection belowground plants did not respond to the same cues or exhibit resulted in VOC emission above ground46. The the same signal-induced response. Communication importance of such responses for fitness of plants via VOC mediated aerial transport was a response to can be explained to some extent, since when a greenhouse whitefly infestation20, whereas CMN attacked, either above or below ground, the whole mediated signal transfer was triggered by an plant is affected and is more susceptible from infection with a pathogen109. Responses of the attacks in all directions. In already mentioned study receiving plants were similar in both cases; receiving by Dicke et al. such transfer can even be directly plants exhibited an increased pathogen resistance, in traced in the plant. Besides the already mentioned the case of mycorrhizal transfer to Alterbaria experiment, where the signal was shown to be solani109, whereas in the case of volatile mediated transmitted through roots using aqueous medium, signalling to pathogen bacteria P. syringae20. Dicke et al. performed two additional experiments Signalling via both described ways simultaneously on different parts of plants. In the first experiment, was prevented by blocking the air flow between an infested leaf was placed in water with a petiole. plants in the case of the experiment on mycorrhizal Subsequently, an unifested leaf was placed in the signalling, and by growing plants in the separate pots same water, resulting in an increased attraction to during the experiment on VOCs aerial signalling. the predatory mites. In the second experiment, a Given that in this system plants were responding to lima bean plant, whose primary leaf was removed in different cues in different experiments, it is possible a way that only the petiole remained, was attached that specific mechanisms of signal emission and to a vial filled with the distilled water. After seven transport are triggered in a way that is dependant up days, water from the vials was collected and healthy on the cue. However signals could also be leaves were placed in it, again resulting in an simultaneously transported via both mediums, or in increased attraction to the predatory mites97. These a time lag, possibly connected with the induction of two experiments, combined with the already the systemic defence response. Furthermore, described root to root signalling one, could indicate induced pathogen resistance of receiving plants in that hydrophilic signals can be transferred through both cases could mean triggering similar or same the whole plant and that such signals can induce a signalling pathways upon perception of the signal. defence response in neighbouring plants at any stage of the transport from an infested leave to These examples open several new questions. First, other leaves or roots. can emitting plants respond to the same above or underground cues using different signalling A good indicator that plants can probably respond to mechanisms (above and below-ground signalling) the same attack with several types of interplant that can be perceived by neighbours, and if so, how signalling, emitted from directly induced as well as are these signals transferred between organs of remote plant organs, are provided by studies on emitting plants? Secondly, are receiving plants able chemical communication and integration of the to perceive different types of signalling through defence signals above and below ground. These different mediums at the same time and if so, how studies present possible mechanisms through which do they integrate and process information? interplant signalling molecules could be transported through plants as well as proof that plants function In order to give a complete answer to the first as whole organisms, integrating and processing question, experiments should be performed where information from all parts simultaneously. different types of signal transport (above and below Below ground interaction of plants with plant ground) are simultaneously observed. Experiments growth-promoting rhizobacteria can prime plant performed so far however have already given some resistance to an attack by herbivores and pathogens insight. As already discussed, separate experiments below as well as above ground. Such induced with the same plant species indicate that same systemic response of plants is at least partially plants can use different types of interplant signalling dependent on JA and ethylene signalling111. Luthe et simultaneously. Furthermore, that plants can emit Plants as whole organisms – integrated view on the interplant communication 18

al. showed an accumulation of defence gene attacked in the leaves and roots, which could result transcripts in maize roots in response to caterpillar in a cross-talk of defence responses to both attacks. above ground feeding and proposed that such They also underlined the importance of systemically defence inducing cues from above ground sent a induced responses for the responsiveness of the signal via the vascular system to the roots, which whole plants on a locally placed attacker and the resulted in an accumulation of defence gene importance of an integration of information from products below ground. Such a mechanism could act roots and shoots by plants for their fitness as a defence mechanism against belowground optimization on a multitrophic level114. Another herbivory as well as a storage mechanism, enabling relevant review, written by Erb et al., focused on the quick translocation of the active defence interactions and integration of arthropod-induced compounds112. That roots are integrated in above and below ground defence responses.They resistance and defence mechanisms triggered by suggest that mediators, such as plant hormones, above-ground herbivory was also shown by Kaplan volatiles or nonhormonal metabolites could be et al. They showed that defence compounds can be transported through plant vasculature ( or synthesized in roots and translocated to the place of phloem) and cell-to-cell signalling115. the attack above ground. Furthermore, plants also Considering all this, we could deduce that emitting use roots as safe storage sites by translocating plants are likely to be able to send signals via nutrients upon above ground attack113. An different pathways and from different parts interesting review on the chemical communication simultaneously. However, practical work has to be between roots and shoots was written by van Dam performed in order to test this and to determine and Bezemer, who showed an integrated view on mechanisms behind it. above and belowground induced plant responses. They pointed out plants can be simultaneously

Fig. 4: Schematic overview on the model of integrated interplant signalling. Emitting plants are subject to either above (A), below (B), or simultaneously above and below ground stress factors, such as herbivory or pathogen infection. Consequently induced stress signals are transported and/or integrated through plant via mediators, resulting in induced systematic response. As part of this defence response, plants emit compounds that can be perceived by neighbouring plants. Such compounds can be emitted either above ground as blends of volatile molecules (VOCs), below ground as root exudates or via common mycotthizal networks (CMNs) or root-root contact. Receiving plants can probably respond to such signals in integrated manner.

Conclusions 19

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