This is the accepted version of the journal article: Ehlers, Bodil K.; Berg, Matty P.; Staudt, Michael; [et al.]. «Plant secondary compounds in soil and their role in belowground species interactions». Trends in Ecology and Evolution, Vol. 35, Issue 8 (August 2020), p. 716-730. DOI 10.1016/j.tree.2020.04.001 This version is available at https://ddd.uab.cat/record/257070 under the terms of the license Manuscript Click here to access/download;Manuscript;PSCbelowgroundinteractionsMai 1 Plant secondary compounds in soil and their role in belowground species interactions 2 Ehlers BK 1 , Berg MP2, 3 , Staudt M4 , Holmstrup M1 , Glasius M5 , Ellers J2 , Tomiolo S1 , Bjerregaard 3 R5 , Slotsbo S1 , and Penuelas J6,7 . 4 5 Full contact details for all authors including institution, city, country, and postcode. 6 1. Department of Bioscience, Aarhus University, Vejlsøvej 25, 8600 Silkeborg, Denmark 7 2. Community and Conservation Ecology Group, Groningen Institute of Evolutionary Life 8 Sciences, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands. 9 3. Department of Ecological Science, Vrije Universiteit Amsterdam, De Boelelaan 1085, 1081 HV 10 Amsterdam, The Netherlands 11 4. CEFE, CNRS, Univ Montpellier, Univ Paul Valéry Montpellier 3, EPHE, IRD, 12 1919 Route de Mende, 34293 Montpellier, France 13 5. Department of Chemistry and Interdisciplinary Nanoscience Center, Langelandsgade 140, 8000 14 Århus, Denmark. 15 6. CSIC, Global Ecology Unit CREAF-CSIC-UAB, 08193 Bellaterra, Catalonia, Spain. 16 7. CREAF, 08193 Cerdanyola del Vallès, Catalonia, Spain 17 18 Twitter handles for authors @SaraTomiolo 19 20 Keywords: Chemical ecology, Soil functioning, Climate change, Soil organisms, Aboveground- 21 belowground interactions 22 23 Abstract 24 25 Knowledge about the effect of plant secondary compounds (PSCs) on belowground interactions in 26 the more diffuse community of species living outside the rhizosphere is sparse compared to what 27 we know about how PSCs affect aboveground interactions. We illustrate here that PSCs from foliar 28 tissue, root exudates, and leaf litter effectively influence such belowground plant-plant, plant- 29 microorganism, and plant-soil invertebrate interactions. Climatic factors can induce PSC production 30 and select for different plant chemical types. Climate change can therefore alter both quantitative 31 and qualitative PSC production, and how these compounds move in the soil. This can change the 1 32 soil chemical environments, with cascading effects on both the ecology and evolution of 33 belowground species interactions, and ultimately soil functioning. 34 35 36 2 37 Plant chemicals mediate species interactions 38 Secondary compounds in plants are major contributors to the chemical diversity of nature. They are 39 formed from primary metabolites (see Glossary) in specific pathways and omnipresent in plants. 40 The distribution of plant secondary compounds (PSCs) is heterogeneous across the plant kingdom, 41 and these compounds exhibit extensive variation both among and within species [1]. Though many 42 PSCs also play pivotal roles in the plant’s primary metabolic processes (such as growth or 43 development), the large majority are known to serve as infochemicals mediating interactions between 44 plants and their biotic and abiotic environments. PSCs can profoundly modify the abiotic environme nt 45 as they are released into the atmosphere, soil, and water of an ecosystem, which in turn determines 46 the community structure of the biotic constituents. For example, PSCs that accumulate in stems and 47 foliage of the vegetation can promote bushfires, that profoundly alter the physicochemical properties 48 of soil and groundwater [2]. Many PSCs are also involved in ecologically crucial interactions for 49 plants, as they can form chemical barriers against pathogens and herbivores, attract pollinators and 50 predators of herbivores, and help disperse fruit and seeds [3]. To fine-tune the release of PSCs, plants 51 “eavesdrop” on their neighbours and increase their own defences when they sense volatiles released 52 from neighbouring plants damaged by leaf herbivores [4]. 53 PSC-mediated interactions between plants and their associated organisms are best-documented for 54 aboveground compounds associated with airborne transport and signalling [3, 5, 6]. PSCs, however, 55 are also omnipresent in the soil, where they mediate interactions with soil organisms and 56 neighbouring plants. Compared to aboveground interactions, these soil PSCs represent a “hidden” 57 link between organisms. Recent reviews of PSCs and belowground interactions [7-9] have focused 58 on PSCs in root exudates and on species interactions, mainly in the rhizosphere. However, less 59 attention has been given to the impact of PSCs on the broader and more diffuse community of 60 associated species further away from roots, which also includes the effects of PSC from foliar tissue 61 and litter decomposition. Climate change may alter both the quantitative and qualitative production 62 of PSCs, which could cascade to affect belowground PSC-mediated community interactions. Given 63 the vital role PSCs play aboveground, we expect PSCs to be equally important to belowground plant - 64 organism interactions. 65 Here, we focus on four major groups of PSCs: terpenes, flavonoids, glucosinolates, and alkaloids 66 (Table 1) that enter the soil matrix. We discuss their origin and fate in the soil and their ecological 3 67 and evolutionary impacts on belowground interactions. Release of PSCs by plants alters soil 68 chemistry, and neighbouring species can either tolerate, adapt to or escape the chemical environme nt 69 this imposes on them. However, the production, release, and transport of PSCs by plants are greatly 70 affected by local conditions, such as microclimate, microbes, herbivore and pathogen pressure. 71 Therefore, changes in climate will affect the quantitative and qualitative production and release of 72 PSCs both directly through changes in temperature and humidity but also indirectly through potential 73 concomitant changes in herbivore and pathogen pressure. As the effects of PSCs on neighbour species 74 are often both compound-specific and dose-specific, there is the potential for cascading effects of 75 PSC-driven alteration of soil chemistry on belowground interactions. So far, however, very little is 76 known regarding such cascading effects in plant-soil community interactions. To encourage more 77 studies on this topic, we outline knowledge gaps and describe best practices for sampling and 78 detecting PSCs in soil, to designate directions for future studies advancing this field of research. 79 80 Origin of PSCs in soils 81 How do PSCs enter the soil? Those formed in roots can be excreted directly into the soil from living 82 and deteriorating tissues [10]. PSCs formed in aboveground organs are deposited into the soil from 83 belowground organs following internal transport [11] or during wet and dry deposition from the 84 atmosphere after volatilization and further chemical reactions [12-14]. PSCs can also be washed from 85 vegetation surfaces with water intercepted during precipitation [15], or they may outgas and leach 86 from fresh and decomposing litter on the soil surface and in the upper horizons [16, 17]. Many plant 87 species accumulate large quantities of PSCs in secretory organs that are either external, such as 88 trichomes, or internal, such as oil blisters or resin ducts, and a strong discontinuous release is expected 89 during the decline and decomposition of these organs. 90 Concentrations of PSCs can be high and persistent in the soil. For instance, mono- and sesquiterpenes 91 released from pine needles, litter and roots are detectable in forest soil throughout the year [18] and 92 can attain high concentrations especially in topsoil but also lower mineral soil, being highest in late 93 summer, and in the winter during snow cover [19]. Glucosinolates, produced by all cruciferous plants, 94 are hydrolysed in the soil to highly bioactive compounds, notably isothiocyanate, nitriles, and 95 thiocyanate. The degradation and hydrolyzation of glucosinolates depend on the presence of the 96 extracellular enzyme myrosinase [20]. Glucosinolates are water-soluble and can more easily be 97 washed out from soil, but isothiocyanates are hydrophobic and adsorb strongly to organic matter [20, 98 21], and may thus persist in the soil for periods of time. The isothiocyanate sinigrin, for example, is 4 99 persistent in the soil of forests invaded by Alliaria petiolata (garlic mustard), where even low 100 concentrations suppress germination and growth of arbuscular mycorrhiza. This suppression disrupt 101 the mycorrhizal association with native plant, negatively affecting the native plant’s resource uptake 102 [22]. The persistence and slow degradation of many PSCs in soil emphasize the ecological and 103 evolutionary potential pressure PSCs can impose on belowground species. 104 105 PSC production under climate change 106 The production of PSCs improves the ability of plants to cope with different abiotic [23] and biotic 107 stressors [5, 6]. Various abiotic factors, such as CO 2 and temperature, affect PSC release by up- or 108 downregulating their production, although the specific PSC compounds that are affected may differ 109 among factors. For example, in northern hemisphere trees, high CO 2 levels generally increase the 110 foliar concentration of phenolic compounds such as flavonoids and tannins, and decrease the 111 production of terpenoid compounds, whereas elevated temperatures lead to the opposite trend [24- 112 26]. Drought can both increase and decrease PSC production dependent on the severity of drought 113 and on the compound. Terpenoids and phenolics in temperate and Mediterranean trees and herbs 114 increased under moderate drought stress but decreased under severe drought [25, 27, 28]. Although 115 severe drought overall decreased total terpenoid emission, some specific mono- and sesquiterpenes 116 either increased or remained unaffected, suggesting an adaptive role of specific compounds in 117 mitigating abiotic stress [27, 28]. 118 119 In addition to environmental effects on the production of specific compounds or compound classes, 120 genetically determined variation in the PSC chemical phenotype (hereafter referred to as chemotype) 121 is present in many species. For example, a strong genetic basis for variation in the identity of leaf 122 chemical compounds has been demonstrated for terpenes produced in oak and pine trees [29] [30, 123 31], aromatic Lamiaceae [32], Asteraceae [33], for glucosinolates produced in the Brassicaceae [34], 124 and for flavonoids in Berberidaceae [35], illustrating that genetic variation for these compounds are 125 present in a wide range of plant families. Thus the (a)biotic environment can shape which compounds 126 are released to the environments not only through differential induction of PSCs, but also by natural 127 selection, favouring genotypes producing specific chemotypes (Figure 1)[36]. Differences in climatic 128 conditions and herbivore pressure favour specific chemotypes both within and among species [29, 129 37, 38]. A recent example of evolutionary changes in PSC production in response to climate change 5 130 is that of wild thyme (Thymus vulgaris), a small shrub widespread in the Mediterranean Basin. The 131 dominant monoterpene in thyme is genetically determined to be a phenolic or a non-phenolic type. 132 These two chemotypes are ecotypes with adaptation to warm dry summers and early winter frost , 133 respectively [39]. The lack of very cold winters in the last 25 years is associated with a significa nt 134 increase in phenolic chemotypes in regions that were previously dominated by non-phenolic types 135 [38]. In addition to differences in their ability to tolerate frost and drought, the toxicities of phenolic 136 and non-phenolic chemotypes differ towards associated plants, microorganisms and herbivores, 137 suggesting that climate-driven changes in the genetic composition of this widespread species may 138 have cascading effects on associated species interactions. 139 A correlation between variation in PSC production and environmental variation holds for many plant 140 species, but we need more long-term experimental studies to understand whether current and future 141 changes in climate and herbivore pressure will result in major induced or genetic changes in PSC 142 production. If so, cascading effects of such changes are expected for belowground species interactio ns 143 and decomposition of organic matter and ultimately nutrient cycling in soil [25, 40]. Moreover, 144 climate change may also affect the concentration of PSCs in soil by altering soil-water content, 145 temperature, and pH. As a result, the levels and circulation of liquid and gas among soil pores (see 146 below) will change and hence the movement and diffusion of PSCs. 147 148 Diffusion of PSC in soil 149 In contrast to atmospheric PSCs, volatile PSCs in the soil can form stable concentration gradients due 150 to the lack of air turbulence. The diffusion of PSCs in soil depends on their physicochemica l 151 properties (Table 1) and the texture of the soil matrix. Fine-textured and clayey soils facilitate the 152 flow of volatile PSCs that are diluted in smaller air volumes due to the smaller pores of these soil 153 types. This generates gradients that are steeper and propagate faster than those in soils with large air 154 spaces. In well-ventilated soils, though, PSCs will rapidly fill large air volumes, albeit at lower 155 concentrations. 156 PSC transfer in soil is also affected by adsorption to the mineral and organic surfaces of soil particles, 157 whose fractions depend on soil type and layer, with A horizons (topsoil) generally containing more 158 organic material than the underlying B horizons (subsoil) [41, 42]. PSCs will adhere to and dissolve 159 on lipo- and hydrophilic surfaces during diffusion, thus forming intermediate pools in equilibr ium 6 160 with the gas-phase concentrations [43, 44]. Soil organisms, therefore, receive PSCs more slowly and 161 with an attenuated amplitude compared to the actual variations in the emission strength of the PSC 162 source. 163 Abiotic factors such as temperature, soil moisture, and pH interfere with the persistence and diffus io n 164 of PSCs in soil. The adsorption of volatile PSCs by the mineral and organic fractions of soil depends 165 on pH, with higher adsorption in alkaline than acidic soils [45]. The acidity of soil-pore water can 166 affect the uptake of volatiles on aqueous surfaces, e.g. by protonating highly reactive sesquiterpenes 167 that are otherwise poorly soluble in water [46]. Periods of rain and drought change the movement 168 patterns of PSCs through soil, with opposite behaviours expected for volatile and non-volatile water- 169 soluble PSCs (Table 1). During rainy weather, volatile PSCs are first concentrated in the soil pores 170 during water penetration and then move with air bubbles to the surface [47], whereas non-volatile 171 water-soluble PSCs are diluted and transported away from their sources [48]. High levels of soil 172 moisture also affect the availability of oxygen for roots and soil microorganisms, eventually 173 generating anaerobic conditions, which influence both plant PSC-producing and microbial PSC- 174 decomposing metabolisms [45, 49]. In contrast, drought may decrease the concentration of volatile 175 PSCs in the soil due to the increase in the volume of air in the soil and better ventilation at the soil 176 surface. Sesquiterpenes, for example, emitted by maize diffuse faster and farther at low moisture 177 levels [50]. 178 Climate change can deeply impact the movement and lifetime of PSCs in soils, either by affecting 179 abiotic drivers directly or by affecting the macro- and micro-biotic communities involved in the 180 liberation, processing and transport of PSCs [51] (see also section “Plant-soil microorga nis m 181 interactions” below). For example, increasing soil temperature will enhance PSC diffusion and 182 exchange rates and alter state and partitioning equilibria between surfaces. Extreme weather events 183 such as thunderstorms and drought events are expected to increase in both frequency and intens ity, 184 which will amplify the asymmetric transport and concentration build-up of polar/non-polar PSCs in 185 soils. Determining the presence, and the fate of PSCs in soil is an under-researched area but is highly 186 relevant to evaluate their ecological and evolutionary impact on the belowground interactio ns 187 discussed below. Understanding the main environmental factors driving these processes is necessary 188 to assess how climate change will alter belowground interactions mediated by PSCs (Figure 1). 189 Different methods may be needed for different types of PSCs, depending on their solubility and 190 volatility (see Box 1, Table 1). 7 191 Ecological and evolutionary impacts of PSCs on belowground species interactions 192 193 Similar to the functions of PSCs aboveground, key organismal interactions in soil, i.e. plant-plant, 194 plant-soil microorganism, and plant-soil invertebrate interactions, are also mediated by PSCs. The 195 identity, concentration, and composition of PSCs belowground play pivotal roles in species 196 interactions and community structuring, and ultimately affect entire ecosystem processes. 197 198 Plant-plant interactions 199 PSCs mediate both competitive and facilitative interactions among plants. Allelopathic effects of 200 PSCs released by a focal plant to the local soil environment can inhibit germination and growth of 201 competitors giving the focal plant a competitive advantage for space and nutrients [52]. The “Novel 202 Weapons” hypothesis [53] is based on the idea that invasive plants release allelochemicals which the 203 native community has not previously encountered and that give the invader a competitive advantage. 204 Allelopathic effects of PSC have helped some invasive plant species to create virtual monocultures 205 in the habitats they invade. For example, production of phytotoxic phenolic acids, and di-, and 206 sesquiterpene lactones likely aided the invasive success of Canadian goldenrod Solidago canadiensis 207 [54, 55] and yellow star thistle Centaurea solstitialis [56]. Allelopathic effects are often less dramatic 208 within the native ranges of plants because coexisting species have evolved a tolerance to local PSCs, 209 or the ability to detoxify them [57]. 210 PSCs released to the soil by one plant species may alter competitive hierarchies among neighbo ur ing 211 plant species both directly when PSC inhibit growth of one species more than another, and indirectly 212 by PSC modifying the soil nutrient availability that favour some species over others. For example, 213 pine soil altered plant competition between spotted knapweed (Centaurea stoebe) and different grass 214 species in favour of grasses. Soil containing pine litter had a higher content of phosphorus (P), and 215 the higher availability of P made the grasses more competitive against Centaurea [58]. Phenolic 216 compounds and terpenes influence soil nitrogen (N) cycling as these compounds inhibit nitrifica tio n 217 and the net N mineralization [59]. This could differentially favour plant species that differ in their 218 preference for nitrate vs ammonium [60]. 219 PSC mediated plant-plant interactions also facilitate neighbour plants via so-called associationa l 220 resistance [61] where neighbour plants benefit from reduced attack by herbivores and pathogens due 8 221 to the deterrent effects of the PSCs released by a focal plant. However, the PSCs in soil may also 222 increase susceptibility to herbivores and pathogens if these plant enemies are attracted rather than 223 repelled by the PSC [52, 61]. How these selective forces are balanced in the multispecies networks 224 present in a natural field needs further study. 225 PSCs can vary among species, genotypes within species, and damaged and healthy tissue, so they 226 contain information about the identity and state of the emitting plant [62]. Plants may therefore use 227 PSCs as a cue to recognize their neighbours and prepare for the competitive encounters by adjusting 228 their competitive growth towards or away from the neighbouring plant [63]. Root exudates and the 229 chemicals they contain have been shown to mediate recognition of neighbour plants [64-66]. The 230 competitive responses to such PSC-mediated neighbour recognition may depend on the history of co- 231 existence between interacting plants [67], and for some plant species also on the genetic relatedness 232 among interacting conspecifics [64, 68]. However, we lack knowledge on the identity of which 233 molecules in root exudates that mediate plant recognition and how plants perceive them. This 234 knowledge is relevant in order to understand if environmental changes in the soil may interfere with 235 plants ability to detect their neighbour. 236 Spatial consistency of PSC production allows PSCs to act as selective agents on other species and 237 favour the genotypes that respond most beneficially to the effects of frequently encountered PSCs. 238 For example, the grass Bromus erectus is a frequent neighbour of T. vulgaris, which releases either 239 phenolic or non-phenolic monoterpenes to the local soil environment. Bromus plants originating from 240 sites where their thyme neighbours produce a non-phenolic monoterpene germinate and grow better 241 in soil rich in non-phenolic thyme monoterpenes compared to similar soil rich in phenolic thyme 242 monoterpenes [69]. Hence, plants can adapt to the chemical environment imposed by frequent 243 neighbours, and this adaptation is highly compound-specific. Changes in the consistency of 244 frequently encountered PSCs could disrupt such adaptive responses. 245 From pairwise plant-plant interaction to communities 246 The PSCs released to the soil from different plant genotypes and species create a mosaic of soil 247 chemical environments. Because different chemical environments favour different species due to the 248 pairwise competitive and facilitative interactions described above, variation in chemical types both 249 within and among populations can contribute to local and regional species richness. For example, the 250 chemical diversity of Scots Pine (Pinus sylvestris) needle terpenes was positively correlated with 251 plant species richness in the vegetation under the trees, and trees with different needle chemistr y 9 252 associated with a different plant species composition [70, 71]. Chemical diversity of T. vulgaris also 253 correlated with plant species richness and composition of the vegetation around thyme plants. Plant 254 species richness was higher, and species composition more similar around thyme plants, than in 255 thyme-free area only few meters away. However, species richness and species composition differed 256 in area with the different thyme chemical types [72]. These examples suggest that the chemical types 257 of dominant plants have an “extended phenotype” [73, 74] where the chemicals released from a focal 258 plant can shape the surrounding vegetation. However, we need more experimental manipula tio n 259 studies investigating the consistency of the effects of variation in PSC released by foundation plants 260 on richness and composition of associated plant communities. These studies will allow us to better 261 understand cause and effect of variation in PSC and to predict the potential cascading effects of 262 qualitative and quantitative changes in PSCs. 263 264 Plant-soil microorganism interactions 265 266 A large proportion of the nutrients present in the soil are not immediately available for plants but 267 require enzymes to degrade compounds into usable parts, and soil microorganisms are major suppliers 268 of these enzymes [80]. In addition, soil symbionts such as mycorrhizal fungi and rhizobacteria are 269 important regulators of plant productivity especially in nutrient poor ecosystems, where they are 270 responsible for 5 to 75% of nitrogen and up to 80% of phosphorous acquired by plants annually [81] 271 The importance and complexity of the interactions between plants and soil microbes have been 272 acknowledged for a long time, but only more recently it has been appreciated that many of these 273 interactions are mediated by PSCs [82]. Given the importance of such interactions, it is likely that 274 plants have evolved strategies to recognize friends from foes and to harness useful partners across 275 distances. For example, flavonoids produced by legumes have a fundamental role in attracting 276 rhizobia and promoting the production of Nod-factor molecules, which are critical for nodulatio n 277 [83]. In this process, both plants and microbes produce chemical signals that allow the plant to 278 recognize the rhizobia as a friend and prevent the activation of immune responses upon initiation of 279 the symbiosis [84]. When rhizobia are not found in the proximity of the rhizosphere and distances 280 between the roots of individual plants and their symbionts are too large to be covered by root 281 exudates, legumes can resort to the help of an intermediary to carry the symbionts to them [83, 85]. 282 The production of volatile PSCs by the legume Medicago truncatula attracts individuals of the free- 283 living nematode Caenorhabditis elegans, which harbours the rhizobium Sinorhizobium meliloti either 10 284 on its cuticle or in its intestine. Isoflavonoids produced by the plant prime the rhizobium to initiate 285 the symbiosis when the nematode is near the roots [75]. 286 Not only can plants affect the composition of specific soil microbial communities via PSC, but they 287 can also alter it when exposed to stress such as herbivore attacks [82]. In many plant species, the 288 presence of pathogens or root feedings fungi corresponds with a shift in the soil microbia l 289 composition close to the rhizosphere. This is a consequence of both the production of specific 290 antimicrobial compounds by the plant rhizosphere and of recruitment of favourable bacteria [86] The 291 blend of PSCs produced by the graminoid Carex arenaria shifts toward a higher concentration of 292 terpenes and terpenoids [59] to attract specific bacteria with antifungal properties when the roots are 293 infected with the pathogenic fungus Fusarium culmorum [87]. Bacteria successfully rallied by the 294 plant can gain from this interaction via the additional nutrients obtained by feeding on the plant 295 pathogens [87]. Recent studies compared wild types of Arabidopsis thaliana with mutants that are 296 unable to synthesize PSCs involved in induced systemic resistance against soil pathogens. 297 Metagenome analysis of root microbiome associated with wild types vs. mutants showed substantia lly 298 different community composition, indicating the important role of PSC in modulating the 299 composition of root microbiome [76]. 300 PSC-induced shifts in the composition of soil microbiota can also be activated by attacks to plant 301 leaves. For example, infestations of white flies (Bemisia tabaci) in pepper plants lead to a change in 302 belowground microflora enhancing anti-pathogen responses [88]. 303 The interactions between plants and soil microorganisms are not unidirectional, as soil microbes can 304 also emit chemical compounds that can be detected and used by plants. For example, secondary 305 compound emissions from soil microflora may help the woodland strawberry, Fragaria vesca, to 306 detect nutrient-rich patches prior to rooting. Stolons can preferentially develop toward substrates 307 emitting unbranched fatty acids and avoid substrates with higher concentrations of terpenes and 308 aldehydes [89]. 309 Many PSCs are used by bacteria and fungi as a source of nutrients [90, 91], and soil microorganis ms 310 can alter and repurpose the PSCs released by plants. In the legume Lotus japonicus, the inocula tio n 311 with its symbiont Mesorhizobium loti leads to a change in the profile of the plant phenolic compounds 312 [60]. The soil bacterial community associated with vetiver, Chrysopogon zizanioides, uses the 313 sesquiterpenes produced by the grass as a source of carbon, and after metabolizing them releases 314 several compounds from vetiver oil [63]. Colonization of roots by arbuscular mycorrhizal fungi can 11 315 increase the production of glucosinolates, phenolic acids, and flavonoids in several plant species [64, 316 65]. 317 On a large scale, the ability of soil microbial communities to process and degrade plant secondary 318 metabolites can have important consequences on plant communities and ecosystem stability. For 319 example, soil microorganisms can either favour or hinder the proliferation on invasive plant species, 320 depending on their ability to degrade the allelopathic compounds produced by invasive plants [92]. 321 A high species-specificity of plant-microbe interactions suggest that single plants can shape their soil 322 bacterial communities, and microorganisms can respond to subtle genetic variation in plants. In 323 cottonwood trees Populus angustifolia, 70% of the variation in soil microbial community 324 composition was explained by plant genotype identity [77]. 325 Although there is still much that we need to understand about the complex interactions between PSC 326 and roots microbiome, progress is being made in elucidating the factors that drive the composition of 327 root inhabiting bacterial communities [78, 79]. Future steps will need to go beyond pairwise 328 interactions between plants and a specific guild of soil microorganism, targeting instead how whole 329 communities of soil microorganisms are affected by PSC and in turn affect plant communities. 330 Climate change may mediate shifts in plant-soil microorganism interactions via shifts in the chemical 331 profile of plants and soil microorganisms. Such studies are highly valuable as the positive effect of 332 certain plant-soil microorganism interactions can be harnessed to either enhance agricultural practices 333 or as biocontrol agents. Understanding how to engineer beneficial soil communities for plant 334 community conservation, sustainable agriculture and preservation of native communities require 335 extensive experimentation and in depth understanding of potential unintended consequences of such 336 manipulations. 337 338 Plant-soil invertebrate interactions 339 Many of the interactions between plants and soil invertebrates are mediated by PSCs, often with 340 significant effects on soil processes that determine soil function and plant productivity. Two major 341 ways can be distinguished through which PSCs mediate the interactions between plants and soil 342 invertebrates. First, soil organisms can be exposed to substantial input of PSCs from aboveground 343 plant tissues, through PSCs leaching from foliar tissue and PSC-rich litter deposition. These PSCs 344 are mostly known as feeding repellents, and many of them with toxic effects to aboveground insect 12 345 herbivores [80], and the available evidence indicates that they also have toxic effects on non-target 346 organisms such as soil invertebrates [81, 82]. Toxic effects can occur if PSCs diffuse across the cuticle 347 or if animals ingest PSC-rich litter, with the main route of exposure determined by cuticle morphology 348 and diet of the soil invertebrate and by the chemical properties of the PSC (see Table 1 for mode of 349 toxicity). Nonpolar (lipophilic) volatile PSCs likely enter animal bodies primarily by simple diffus io n 350 across the cuticle and easily partition into cellular membranes [81]. High concentrations of nonpolar 351 PSCs can occur regularly in soil, especially in older litter and near roots, as is the case for terpenoids 352 (1-5 mg g-1 fresh weight) [2]. The likelihood of exposure is increased because terpenoids will remain 353 in the litter layer for several months until fully decomposed [83]. Exposure to polar PSCs is mainly 354 through ingestion of litter or roots, for example in case of isothiocyanates. Isothiocyanates have a 355 high toxicity towards non-target soil invertebrates, including beneficial macro-detritivores that 356 promote nutrient cycling and soil fertility [84]. Currently, the relative importance of the two exposure 357 routes, either via contact with volatile or water dissolved PSCs in pore water and diffusion across the 358 cuticula or via ingestion of PSC-rich litters to soil fauna and how this affects soil fauna functio na l 359 community composition is poorly known. 360 An unexplored aspect of the lipophilic PSCs is that their spread can be facilitated by animal transport. 361 Soil animals can accumulate high internal concentrations of these PSCs in the vicinity of roots and 362 litter, and subsequently, deliver them to other soil microenvironments with lower chemical activity 363 when they move away from the PSC source. Aquatic Protozoa have demonstrated such anima l- 364 facilitated transport [85], but more research is needed to explore this phenomenon mediated by soil 365 animals. In order to explore the toxicity of PSCs to soil invertebrates under field conditions, more 366 precise analytical determinations of concentrations of PSCs and their degradation products in the 367 environment are needed. This necessitates new experimental systems of exposure, e.g. by using the 368 framework of chemical activity for volatile compounds [81]. 369 The second way in which PSCs can mediate plant invertebrate interactions in the soil is by targeted 370 release of PSCs from the roots. These PSCs can attract or deter soil invertebrates and serve as 371 chemical signals released by the plant to control trophic interactions with plant-parasitic nematodes, 372 root herbivores, and their predators. The use of PSCs in tri-trophic interactions between plants and 373 belowground organisms may be similar to the extensively studied chemically mediated interactio ns 374 between plants, foliar herbivores, and their natural aboveground predators. Attack by root herbivores 375 has been shown to induce a change in the quality and quantity of PSCs released by plant roots, which 376 can then function as a signal to attract soil-dwelling predators and reduce the pest population [9, 86, 13 377 87]. Entomopathogenic nematodes often function as indirect defences for plants [88, 89]. For 378 example, larvae of the longhorn beetle Tetraopsis tetraophthalmus feed on the roots of the common 379 milkweed Asclepias syriaca which releases volatiles into the soil that attract entomopathoge nic 380 nematodes, preventing the loss of plant biomass to herbivory [90]. Maize roots increase sesquiterpene 381 exudation when attacked by insects, thus attracting entomopathogenic nematodes that feed on root 382 herbivores [91]. However, notwithstanding this potential protective role of PSCs via attraction of 383 beneficial nematodes, these PSCs can also attract the root herbivores themselves. The net effect of 384 the attraction of both advantageous as well as detrimental nematodes on plant fitness has to be 385 evaluated. Most examples of tri-trophic interactions belowground involve entomopathoge nic 386 nematodes, but one of the few papers that reports on other soil fauna in tri-trophic interactions is a 387 case in which tulip bulbs infested by the rust mite Aceria tulipae, emits volatile signals to the 388 predatory mite Neoseiulus cucumeris [92]. Given the importance of chemical cues in belowground 389 signalling, the effect of PSCs on tri-trophic interactions is expected to be omnipresent, but currently, 390 we do not know enough about the spatial dimensions of volatile and water-dissolved PSCs effects on 391 plant-root herbivore or parasite-herbivore interactions [93]. Abiotic gradients in soils are rather short 392 and steep compared to aboveground systems, but currently, we are ignorant of the spatial dimens io ns 393 of volatile and water-dissolved PSCs effects on plant-root herbivore or parasite-predator interactio ns. 394 395 PSCs and belowground co-evolutionary interactions 396 The examples above demonstrate the different vital roles that PSCs play in belowground plant-plant, 397 plant-soil microorganism, and plant-soil invertebrate interactions. Some of these interactions are 398 examples of the evolutionary adaptation of species to frequently encountered PSCs. Except for 399 interactions specific to the rhizosphere, no studies have yet clearly shown whether adaptations of 400 organisms to specific PSCs reciprocally alter PSC composition and production in plants, i.e. if PSC- 401 mediated belowground interactions result in pairwise or diffuse co-evolution. Understanding if 402 belowground interactions are a source of selection on the chemical variation of PSC-producing plants 403 is not straightforward because of the unspecific nature of the interactions. Belowground interactio ns 404 often consist of multiple interactions, therefore, estimating the consequences for fitness in PSC- 405 producing plants is difficult. Also, for PSCs entering the soil via leaf leachates, the main selective 406 force determining which PSCs are produced and in what concentration may largely depend on 407 aboveground biotic interactions and local climate. In addition, the multifunctionality of PSCs extends 14 408 beyond biotic interactions, which is a further complication in the evaluation of fitness consequences 409 in PSC-producing plants. For instance, maize produces benzoxazinoid compounds not only as a 410 defence against generalist herbivores but also as chelating agents to acquire iron as a micronutrie nt. 411 The larvae of western corn rootworm Diabrotica virgifera, are not injured by benzoxazinoids, but 412 this specialist herbivore instead takes advantage of the presence of complexes between iron and these 413 PSCs not only to detect the plant but also forage on these complexes [94]. 414 Climate-induced shifts in the release of PSCs into the soil (Figure 1) may alter the outcome of within- 415 and between-species interactions in ways that we still cannot predict. Long-term studies and the 416 development of ecometabolomics [95] for elucidating the responses of PSC changes in plant 417 communities due to warming and altered precipitation from climate change are particularly warranted 418 for a better understanding of, and ability to predict, these shifts in PSCs production, diffusion in soil, 419 and effects on belowground interactions. 420 421 Concluding remarks and perspectives 422 Soil is a theatre of facilitation, symbiosis and warfare deployed by plants and the various organisms 423 living in it, and PSCs plays a major role mediating many of these interactions. Plants and soil 424 organisms have adapted to withstand, detoxify or use the cocktail of PSCs originally meant to harm 425 some of them. Understanding PSC mediated relationships at community scale and identifying the 426 compounds involved in these interactions is therefore important for better insight into the functio ning 427 of these systems and their evolution, especially in changing environments. High-throughp ut 428 technologies are increasingly becoming available and may help to identify and determine the 429 concentrations, transport and modes of action of PSCs in soils. We encourage the creation of open, 430 community-wide, curated, labelled, broad-spectrum PSC data sets across plant species and soils, as 431 this would greatly increase the transfer of knowledge between scientist studying plants, microbes, 432 and invertebrates in this biological belowground theatre. This could be a platform similar to, e.g. the 433 TRY plant trait data base (http://www.try-db.org) or PSC data sets could be added and incorporated 434 to such established well-functioning platforms. The warranted study of the quantitative and 435 qualitative effects of climate change on PSCs and their interactions with the environment and 436 organisms would also benefit from these PSC data sets. The alterations of the production of PSCs 437 due to climate change are highly variable, depending on dose, timing, PSC, and species. These 438 changes in the production of PSCs can lead to unforeseen consequences for soil structure and functio n 15 439 and can disturb biological feedbacks on soil chemistry and biology, perhaps even on atmospheric 440 chemistry and climate [96], with a direction and intensity that warrants in-depth investigation. 16 441 References 442 443 1. Moore, B.D. et al. (2014) Explaining intraspecific diversity in plant secondary metabolites in an 444 ecological context. New Phytol. 201, 733-750. 445 2. Alessio, G.A. et al. (2008) Implications of foliar terpene content and hydration on leaf 446 flammability of Quercus ilex and Pinus halepensis. 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Spietelun, A. et al. (2010) Current trends in solid-phase microextraction (SPME) fibre 667 coatings. Chem. Soc. Rev. 39, 4524-4537. 668 111. Ferreira, A.M.C. et al. (2011) Stir bar sorptive extraction of parabens, triclosan and methyl 669 triclosan from soil, sediment and sludge with in situ derivatization and determination by gas 670 chromatography–mass spectrometry. J. Chromatogr. A 1218, 3837-3844. 671 112. EPA, Closed system purge-and-trap extraction for volatile organics in soil and waste samples., 672 US Environmental Protection Agency, 1996, pp. 1-24. 673 674 675 21 676 BOX 1. Sampling of PSCs for identification and quantification. 677 The heterogeneous chemical and physical nature of soil creates spatial differences that complicate 678 representative sampling. In addition, PSCs possess a high diversity of functional groups, volatilities, 679 and polarities. Their belowground presence can thus be in air, in pore water, or adsorbed to solid 680 matter. Representative sampling may therefore require a combination of sampling techniques. 681 Sampling techniques for PSCs in soil include extraction (solvent or solid phase), head-space 682 analysis, and purge and trap techniques [7]. 683 Solvent extraction relies on the principle of “like dissolves like”. Polar compounds are thus 684 extracted in polar solvents (e.g. water, methanol) and nonpolar compounds in nonpolar solvents 685 (e.g. n-hexane, dichloromethane). Several hundreds of grams of soil, however, may be needed for 686 the representative sampling of PSCs with low concentrations in soil [109]. Dilution beyond 687 detection limits may occur, so subsequent sample concentration by solid-phase extraction (SPE) or 688 evaporation (with the risk of loss of volatiles) may be necessary. Emission of PSCs directly from roots 689 have been investigated using primarily solid-phase micro extraction (SPME) or adsorption tubes (with e.g. 690 Tenax) coupled with GC-MS analysis in a number of studies as reviewed in [7]. 691 Solid-phase micro extraction (SPME), head-space analysis, and purge and trap methods avoid the 692 dilution effect of solvents but are more selective in the compounds sampled. SPME relies on the 693 equilibrium between a fibre coated with a sorbing material and most often the gas phase (potentially 694 also water or solid phase). This technique allows in-situ sampling for evaluating small-scale spatial 695 differences. The sorbing material determines the selectivity of the compounds sampled [110], 696 whereas soil type, water content, and pH influence the equilibrium. Stir-bar sorptive extraction 697 (SBSE), where a magnetic rod is coated with sorbing material, may provide up to 1000-fold higher 698 sensitivity but requires thermal desorption for subsequent instrument introduction. SBSE is useful 699 for extraction from water, as demonstrated for many organic pollutants [111]. Identifying the mode 700 of transport of PSCs (i.e. in gas or water phase) could be accomplished by sampling the soil gas 701 phase using SPME and subsequently washing the soil and extracting using SBSE. 702 Collecting only head spaces using an air-tight syringe is a potential way to sample only volatile 703 PSCs (i.e. VOCs). The concentration of VOCs from soil, however, may be <10 g m-3 , which is 704 below the detection limit for head-space analysis. This limitation may be overcome with purge and 705 trap methods recommended for analysis when concentrations are <200 g kg-1 [112], which have 706 recently been applied for sampling isoprene, monoterpenes, and sesquiterpenes in forest soil [19]. 22 707 Glossary box: 708 709 Allelochemical is a compound produced by an organism that can have a detrimental physiological 710 effect on individuals of another species when released into the environment. 711 Allelopathic effect is the interference effect of chemical compounds released by a plant on the 712 growth and performance of other plants. An allelopathic effect usually implies a direct negative 713 (harmful) effect, but some definitions also include positive effects. 714 Narcotic effect refers to the unspecific effects of lipophilic organic chemicals on membrane 715 fluidity. These chemicals tend to partition into the lipid compartments of the organism, causing 716 (nonpolar) narcosis or baseline toxicity. 717 Phytotoxic refers to chemical compounds toxic to plants, including any adverse effect on plants 718 such as delayed seed germination, inhibition of growth, death, or destruction of plant tissue. 719 Plant secondary compounds (PSCs) are organic compounds typically formed from primary 720 metabolites in specific pathways. Some PSCs are involved in plant’s primary metabolic processes, 721 but the large majority of these are known to serve as infochemicals mediating interactions and are 722 important for plants to survive in the environment, e.g. by their repellent, attractant, or toxic effects 723 on other organisms. 724 Primary metabolites are compounds from metabolic pathways present in all plants and are 725 essential for growth, development, and reproduction. These metabolites comprise amino acids, 726 nucleic acids, peptides, various carbohydrates, and lipids. 727 Rhizosphere is the microecological zone of soil in direct proximity to plant roots and includes the 728 organisms in that soil that are directly affected by root exudates. 729 730 731 732 733 23 734 Legend to Figure 1. Direct and indirect effects of climatic conditions (red box) and aboveground 735 herbivore pressure (green box) on plant secondary compound production (yellow boxes) and input 736 into the soil, and its belowground interactions (brown box). In addition, climate affects how PSCs 737 are transported and degrade in soil. Dashed arrows and boxes indicate processes and effects that are 738 less well understood and are elucidated in the main text. 739 740 741 742 743 744 745 746 747 748 749 750 751 752 753 754 755 756 757 24 758 Table 1. Overview of the important classes of PSCs involved in mediating belowground 759 interactions, and best practices for sampling and analysing them. The chemical properties of PSCs 760 affect their position and transport in the soil matrix, with polar PSCs dissolved in water, volatile 761 PSCs diffusing through air spaces in the soil, and less polar non-volatile PSCs adsorbed to the 762 organic fraction of soil. 763 25 Compound Example of Polarity Water Volatility Ecological interactions/ The analytical instrument for detection class structure solubility impacts and effects on associated soil organisms in soil (see also Box 1) Terpenes Low Low High Defensive and signalling Monoterpenes and highly volatile sesquiterpenes: S compounds, many with a Synthesized characteristic odour (e.g. - with PDMS adsorbent, purge-and-trap with Tenax from isoprene pinene from pine, limonene units (C5 H8). adsorbent and subsequent GC-MS [19], [99], from citrus). Antioxidant, Includes antimicrobial, and On-line measurement with Proton-transfer-reaction monoterpenes phytotoxic properties [3]. MS [100]. (C10 H16 ) and Toxic to invertebrates due sesquiterpenes Nonpolar sesquiterpenes likely adsorbed to soil to baseline toxicity (C15 H24 ). -Pinene (“narcotic” effect) via particles require extraction with organic solvents an interaction with cellular membranes [97]. Involved evaporation followed by GC-MS [41]. in formation of aerosol particles, thus affecting climate [98]. Flavonoids Low to Low to Low Both defensive and Most likely present belowground in water and ad intermediate intermediate signalling; promote Extraction using polar solvents (water, methanol) General symbiosis (used by legumes metals are formed [104]. structural C-15 Presence of to attract rhizobia), various skeleton sugar Extracts can be analysed with reversed-phase LC-M antimicrobial activities [101, consisting of moieties 102]. Can interact with Ca 2+ two phenyl rings increases 26 and a Basic flavonoid solubility in channels in cell membranes Aglycones may be sampled using SPE [101] and ar structure heterocyclic ring water causing cytotoxic influx of to GC-MS after derivatization. Greater sensitivity m (C6-C3-C6). Ca2+ [103]. obtained with SBSE and subsequent GC-MS. Glucosinolates High High Low Due to polarity and water Mostly present in the water phase due to high pol solubility, are readily taken Extraction using polar solvents (water, methanol) Derived from up by soil organisms, where with LC-MS [17] or GC-MS after derivatizati glucose and an they can be converted to concentration in the sample. amino acid. highly reactive compounds Most are found Sinigrin such as isothiocyanate and in the order thiocyanates. Toxicity Brassicales (30). pathways include inhibition of cytochrome P 450 detoxification complex, induction of programmed cell death (apoptosis), and genotoxic effects [106]. Alkaloids Low to Low to Low Mostly defensive Low concentration in soil, so clean-up and pre intermediate intermediate compounds. Phytotoxic, necessary. Polarity can be adjusted with pH changes Highly diverse, toxic (e.g. strychnine), and with liquid-liquid extraction, SPE, or SPME [108] usually with a narcotic effects (e.g. both LC-MS and GC-MS. basic nitrogen morphine, codeine). Can atom either in interfere with gene Nicotine 27 cyclic rings or transcription and protein open chains. synthesis [107]. 764 765 28 Outstanding Questions Outstanding Questions To what extent does the plant secondary compound (PSC) plume originally released into a soil compartment become chemically degraded during diffusion and mixed with secondarily formed and other ambient PSCs? Does this dilution and mixing with other PSCs impair the signal quality for the receiving soil organisms or have they adapted to and even benefited from signal noise, e.g. to improve their foraging efficiency? How will climate change affect PSC production in both above- and belowground organs and how will these effects in turn affect the belowground interactions of associated communities of microbes, plants, and soil invertebrates? What is the relative importance of specific PSCs for both plant-soil invertebrate and plant-plant interactions and can we understand their prevalence from knowledge on their mode of action? If the effects of PSC on associated species is very compounds specific, quantitative and qualitative changes in the input of PSC to the soil can have cascading effects on richness and composition of belowground soil communities that may ultimately alter soil functioning. What is the importance of a common co-evolutionary history between PSCs in soil and the organis ms exposed to them for shaping the outcome of their interactions? How important are belowground interactions in determining the composition and concentration of PSCs released by the producer? In particular, what is the impact of selection (if any) that belowground interactions can exert on PSC composition and production in leaves relative to aboveground interactions and climate? Highlights HIGHLIGHTS: - Plants produce a high diversity of secondary compounds that are released to their surroundings. - The role of plant secondary compounds (PSCs) on aboveground interactions has been well studied, but less is known about the role of PSCs in mediating and determining the outcome of belowground interactions – especially interactions beyond the rhizosphere. - PSCs enter the soil directly by excretion from belowground plant organs or indirectly by washing from the canopy and leaching from litter. - Leaching of PSCs creates a heterogeneous soil chemical environment and mediate plant-plant interactions, plant-microbiome associations, and plant-soil invertebrate relationships. This affects and shape the diffuse community of species living in soil also further away from the rhizosphere. - Climate change can substantially alter the quantitative and qualitative production of PSCs released to the soil, which may change belowground species interactions and affect species composition, richness, and ultimately soil functioning. Figure