Emerging role of roots in plant responses to aboveground insect herbivory

Insect Science (2013) 20, 286 296, DOI 10.1111/1744-7917.12004 INVITED REVIEW Emerging role of roots in plant responses to aboveground insect herbivory Vamsi J. Nalam 1, Jyoti Shah 2 and Punya Nachappa 1 1 Department of Biology, Indiana University-Purdue University, Fort Wayne, Indiana and 2 Department of Biological Sciences and Center for Plant Lipid Research, University of North Texas, Denton, Texas, United States Abstract Plants have evolved complex biochemical mechanisms to counter threats from insect herbivory. Recent research has revealed an important role of roots in plant responses to above ground herbivory (AGH). The involvement of roots is integral to plant resistance and tolerance mechanisms. Roots not only play an active role in plant defenses by acting as sites for biosynthesis of various toxins and but also contribute to tolerance by storing photoassimilates to enable future regrowth. The interaction of roots with beneficial soilborne microorganisms also influences the outcome of the interaction between plant and insect herbivores. Shoot-to-root communication signals are critical for plant response to AGH. A better understanding of the role of roots in plant response to AGH is essential in order to develop a comprehensive picture of plant-insect interactions. Here, we summarize the current status of research on the role of roots in plant response to AGH and also discuss possible signals involved in shoot-to-root communication. Key words jasmonic acid, secondary metabolites, shoot-to-root communication, soilborne microorganisms Introduction Current understanding of plant insect interactions is drawn largely from the response of plant foliar tissue to insect herbivory (Howe & Jander, 2008; Wu & Baldwin, 2010). Plants combat insect feeding using both constitutive defenses and defenses that are induced only in response to attack (Karban & Baldwin, 1997). Inducible defenses can be classified as either direct or indirect. Direct defenses affect the biology of the attacker directly, whereas indirect defenses, which are more relevant in defense against herbivores, affect the herbivore by attracting its natural enemies (Karban & Baldwin, 1997). Inducible defenses offer several advantages compared to constitutive defenses, such as reduced cost and increased vari- Correspondence: Punya Nachappa, Department of Biology, Indiana University-Purdue University, Fort Wayne, Indiana 46805, USA. email: nachappa@ipfw.edu ability in the plant phenotype, resulting in increased efficiency of the inducible defense (Karban & Baldwin, 1997; Zangerl, 2002; Cipollini et al., 2003). Given that all plant tissues (leaves, roots, stems, flowers, and fruits) are consumed by insect herbivores, our understanding of plant insect interactions is skewed. In several plant species, the biomass of roots is far greater than that of the shoots; hence, roots provide an incredibly attractive resource to various soil-dwelling insect pests. And indeed, herbivory of roots by belowground herbivores (BGH) results in substantial damage to plant roots and significantly impacts overall plant fitness (Blossey & Hunt-Joshi, 2003). Studies examining plant responses to BGH have revealed that similar to aboveground responses, roots also employ direct and indirect induced defenses (Rasmann & Agrawal, 2008; van Dam, 2009; Erb et al., 2012). The activation of defenses due to insect herbivory either belowground or aboveground often results in the induction of defenses systemically throughout the plant. Systemic defenses can thereby influence the outcome of not only plant insect C 2012 Institute of Zoology, Chinese Academy of Sciences 286

Root responses to aboveground herbivory 287 interactions but also link the belowground and aboveground biota (reviewed in Bezemer & van Dam, 2005; Johnson et al., 2008). In terrestrial plants, roots serve many different purposes including, absorption of water and nutrients, anchoring the plant to soil, and storage of photoassimilates (Öpik et al., 2005). Roots are also increasingly being recognized as active participants in plant responses to AGH (Erb, 2012). The earliest report that root-derived secondary metabolites are involved in plant defenses against aboveground herbivores (AGH) was made as early as 1941 when it was shown that the alkaloid nicotine was primarily synthesized in the roots of tobacco plants (Dawson, 1941). Of late, there has been a surge of interest in elucidating the contribution of roots in shoot defenses against insect herbivores (Kaplan et al., 2008b; Rasmann & Agrawal, 2008; Erb et al., 2009b; Erb et al., 2012). For example, a recent study shows that root-derived oxylipins contribute to host susceptibility by promoting population growth rate of an aboveground insect herbivore. Feeding by the generalist aphid (Myzus persicae) resulted in the induction of expression of a 9-lipoxygenase encoding LIPOXYGE- NASE5 (LOX5) gene and a corresponding increase in the LOX5-synthesized oxylipins, 9-hydroperoxy, and 9- hydroxy-fatty acids only in the roots of aphid-infested Arabidopsis plants. These oxylipins were translocated to the shoot, where they promoted aphid feeding, body water content and fecundity (Nalam et al., 2012). Studies such as these and others highlight the need to include roots to develop a better understanding of plant insect interactions. In this review, we will focus on recent advances in understanding (i) the contribution of roots to plant defenses against AGH and (ii) the identity of potential signal(s) involved in integrating shoot and root responses to AGH. Contribution of roots to defense against AGH Root-derived defenses Plants synthesize a variety of secondary metabolites to withstand insect attack. While some of these secondary metabolites are toxic to the herbivore, others reduce the palatability of the plant to the insect. Alkaloids and terpenoids, which are among the most metabolically diverse classes of secondary metabolites, represent a major class of insecticidal metabolites (Facchini, 2001; Aharoni et al., 2006; Ziegler & Facchini, 2008). Others include glucosinolates, saponins, tannins, furanocoumarins, and cyanogenic glycosides (Wu & Baldwin, 2010; Yamane et al., 2010). In addition to secondary metabolites, a broad array of proteins that are induced in response to herbivory are also involved in plant defense responses (Zhu-Salzman & Liu, 2011). The elucidation of the biosynthetic pathways for some of these metabolites and induced proteins highlight roots as an important site of synthesis (Table 1; reviewed in van der Putten et al., 2001; Rasmann & Agrawal, 2008; Erb et al., 2009b; Erb, 2012). The most well studied insecticidal secondary metabolite is nicotine, a neuroactive compound synthesized in large amounts by plants of the genus Nicotiana. Nicotine is synthesized in roots from where it is translocated to shoots and stored in vacuoles of leaf cells, thus providing a constitutive defense against insects (Dawson, 1941; Morita et al., 2009). In response to herbivory or methyl jasmonate treatment, the accumulation of nicotine increased in roots and lead to increased concentrations in shoots and thus enhanced protection against insect herbivory (Baldwin et al., 1994). The synthesis of alkaloids in roots is not limited to plants of the genus Nicotiana. A similar pattern is observed for the synthesis of tropane alkaloids that may be involved in leaf defense in the Solanaceae family of plants (Ziegler & Facchini, 2008). In some instances, the precursors for secondary metabolites that participate in shoot defense are synthesized in roots and then transported to shoots where they undergo further modifications. For example, the precursor for pyrrolizidine alkaloids, senecionine N-oxide is produced in the roots and then translocated throughout the plant tissue (Toppel et al., 1987; Hartmann & Ober, 2000). A second example is umelliferone, which is synthesized in significant quantities in roots of the bishopweed (Ammi majus) (Sidwa-Gorycka et al., 2003). Umelliferone is the precursor for several furanocoumarins that act as feeding deterrents for insects and have antifungal and antibacterial properties (Berenbaum, 1978; Yamane et al., 2010). Another class of compounds, the glucosinolates found mainly in plants belonging to Brassicaceae, function in defense against herbivores and pathogens. In black mustard (Brassica nigra), foliar herbivory by the larvae of cabbage butterfly (Pieris brassicae) resulted in the accumulation of higher levels of indole glucosinolates in the roots (Soler et al., 2009). An increase in the levels of indole glucosinolates was also observed in roots of field mustard (Brassica campestris), when shoots were treated with the defense elicitors salicylic acid (SA) and jasmonic acid (JA; Ludwig-Müller et al., 1997). However, since glucosinolates can be readily loaded and transported through the phloem (Chen et al., 2001), it is as yet unclear whether these compounds are synthesized exclusively in the roots or whether they are synthesized in infested shoots and then transported systemically throughout the plant including the root.

288 V. J. Nalam et al. Table 1 Insecticidal factors synthesized in roots in response to AGH. Increase/ Influence Plant Induction by Altered root defense decrease of on Reference root defense AGH Brassica nigra Pieris brassicae Indole glucosinolates Increase Yes Soler et al., 2009 Zea mays Spodoptera frugiperda Mir1-CP Increase Yes Lopez et al., 2007 Arabidopsis thaliana Myzus persicae 9-LOX derived oxylipins Increase Yes Nalam et al., 2012 Senecio jacobea Mamestra brassicae Pyrrolizidone alkaloid Decrease Yes Hol et al., 2004 Gossypium herbaceum Spodoptera exigua Terpenoid aldehydes Decrease NA Bezemer et al., 2004 Brassica campestris JA, SA Indole glucosinolates Increase NA Ludwig-Müller et al., 1997 Abelmoschus esculentus SA PR proteins Increase NA Nandi et al., 2003 Nicotiana attenuata MJ, MD Trypsin proteinase Increase NA van Dam et al., 2001 inhibitors Nicotiana sylvestris MD Nicotine Increase NA Baldwin et al., 1994 Cynoglossum offininale MD Pyrrolizidone alkaloid Increase NA van Dam & Vrieling, 1994 Secale cereale MD Hydroxamic acids Increase NA Collantes et al., 1999 MD, mechanical damage; MJ, methyl jasmonate; SA, salicylic acid; JA, jasmonic acid; PR, pathogenesis proteins; NA, not applicable. Arthropod-inducible proteins (AIPs) also provide a broad spectrum of resistance in several plant species by providing postingesting plant defenses (Zhu-Salzman & Liu, 2011). In the case of one such AIP, the levels of Maize insect resistance 1-cysteine protease (Mir1-CP) increased in the leaf tissue of a resistant maize (Zea mays) genotype (Mp708) in response to foliar herbivory by the larvae of fall armyworm (Spodoptera frugiperda) (Lopez et al., 2007). Mir1-CP accumulated in root xylem tissue 24 h after foliar feeding and was also found in the xylem tissue of leaves. Furthermore, removal of roots prior to larval feeding prevented the accumulation of Mir1-Cp in leaves, suggesting that the protein is first synthesized in the roots and transported to the leaf tissue through the vasculature. The synthesis of defensive factors by roots in response to foliar herbivory is not a universal phenomenon. In certain instances, there is a reduction in the levels of plant toxins that are normally synthesized in roots. For example, the levels of pyrrolizide alkaloids, which are produced in roots (Toppel et al., 1987) decreased in the roots of ragwort (Senecio jacobea) in response to AGH by the cabbage moth (Mamestra brassicae) (Hol et al., 2004). In other cases, defensive compounds normally induced in roots in response to BGH are not induced during AGH. For instance, belowground herbivory by wireworms (Agriotes lineatus) resulted in increased accumulation of terpenoids in both roots and shoots. However, a similar increase in terpenoid content was observed only in shoots during aboveground herbivory by beet armyworm (Spodoptera exigua) (Bezemer et al., 2003, 2004). This observation suggests that although BGH may change the level and distribution of defensive secondary metabolites, the same may not hold true for all cases of AGH. One possibility to explain the suppression of root-induced defense in these cases is that herbivores themselves manipulate host responses to suit their needs by either suppressing host defenses or activating alternate mechanisms that promote the AGH. Roots limit resource availability to AGH A major function of plant roots in many species is the storage of food and nutrients. Roots can potentially contribute to the plants ability to tolerate AGH by acting as storage sites for photoassimilates. For instance, in response to AGH, photoassimilates are translocated to the roots making them inaccessible to the herbivore. The reallocation of stored photoassimilates aboveground can then occur after AGH pressure has reduced, thus enabling aboveground growth and reproduction to resume. There are several lines of evidence that indicate that foliar herbivory, shoot hormone applications or mechanical damage can result in reallocation to storage tissue (reviewed in Orians et al., 2011). In maize for instance, foliar insect herbivory by grasshoppers resulted in the mobilization of photoassimilates to roots (Holland et al., 1996). A similar process occurs in Nicotiana tabaccum after AGH by tobacco hornworm (Manduca sexta) larvae (Kaplan et al., 2008a). In Populus, treatment of the shoot with the defense hormone JA or AGH by Gypsy moth (Lymantria

Root responses to aboveground herbivory 289 dispar) larvae resulted in the increased transport of leaf photosynthate to the stems and roots within hours of treatment (Babst et al., 2005; Babst et al., 2008). In tomato (Solanum lycopersicum), AGH by tobacco hornworm (M. sexta) resulted in a significant increase in the concentrations of various sugars, sugar alcohols and organic acids in the roots (Steinbrenner et al., 2011). To achieve an increase in the transport of photosynthate to the roots, plants can either increase loading into phloem and/or increase unloading into the roots (Turgeon & Wolf, 2009). The increased carbon sink strength of the roots can be achieved in part by an increase in the activity of invertases, a sugar-cleaving enzyme (Roitsch & González, 2004). Indeed, an increase in invertase activity was found in roots in response to AGH by the tobacco hornworm (Kaplan et al., 2008a). Insect-derived elicitors present in the regurgitant of chewing insects are also capable of inducing resource reallocation to roots (Schwachtje et al., 2006). The application of M. sexta regurgitant to leaves of tomato plants resulted in increased allocation of carbon to the roots (Gómez et al., 2012). This herbivoreinduced resource-reallocation is thought to be regulated by SNF1-related kinases, which play a central role in cell energy metabolism (Halford & Hey, 2009). In Nicotiana attenuata, the transcript levels of SNF1-related kinases were rapidly downregulated in leaves treated with insectderived elicitors resulting in increased assimilates transported to roots (Schwachtje et al., 2006). Although the regulatory mechanisms that initiate and control reallocation are not fully understood (Erb et al., 2009a; Erb, 2012), it is clear that roots play an active role in limiting resource availability to AGH. Interaction of roots with beneficial soil-microbes influences AGH The interaction of roots with beneficial soil-borne microbes also influences the outcome of the aboveground interactions between the plant and herbivore. The association of roots with certain plant growth-promoting rhizobacteria and mycorrhizal fungi not only results in the promotion of plant growth but also the induction of resistance against a wide variety of AGH and pathogens. This phenomenon termed induced systemic resistance (ISR) provides protection against a wide range of diseases (van Oosten et al., 2008; Doornbos et al., 2010). Although, several different beneficial soil-borne microorganisms can induce ISR, the mechanism of induction of ISR follows similar patterns and is mediated by JA or jasmonic ethylene (ET) sensitive pathways which are commonly involved in plant defense responses (van Oosten et al., 2008; Doornbos et al., 2010). Beneficial soil-borne microorganisms can therefore indirectly influence with outcome of plant interaction with an AGH via plant-mediated mechanisms. There are examples of both positive and negative effects of such interactions (reviewed in Pineda et al., 2010). For instance, the association of Acremonium alternatum Gams (Ascomycotina), an endophytic fungus with the roots of cabbage (Brassica oleracea) resulted in increased mortality and reduced growth rate of the surviving larvae of the diamond back moth (Plutella xylostella) (Raps & Vidal, 1998). The association of a plant-growth promoting rhizobacteria, Bacillus amyloquefaciens, with tomato or sweet pepper (Capsicum annuum) roots conferred protection against phloem-feeding insects, the green peach aphid (M. persicae) and silverleaf whiteflies (Bemisia argentifolii)(murphy et al., 2000; Herman et al., 2008). In these cases it is plausible that root association with the beneficial soil-microbes resulted in the activation of ISR. In a more recent study, the role of ISR was demonstrated in the association of Bacillus subtillis with the roots of tomato plants, which retarded silverleaf whitefly development (Valenzuela-Soto et al., 2010). By comparison, the association of Rhizobium leguminosarum with white clover (Trifolium repens) resulted in a positive effect on the Egyptian cotton leafworm (Spodoptera littoralis) and a neutral effect on the green peach aphid (Kempel et al., 2009). The species or the strain of the beneficial soil-borne microorganisms also impacts the outcome of plant interaction with a specific AGH. For instance, in rice (Oryza sativa) a combination of different Pseudomonas fluorescens strains had a stronger impact as compared to the influence of the strains individually (Saravanakumar et al., 2007). The impact of root-colonizing microbes on AGH can also be influenced by the degree of specialization of the herbivore. Studies on Arabidopsis show that association of roots with P. fluorescens results in the activation of JA/ET responsive defense pathways which do not affect feeding by the specialist caterpillar, Pieris rapae. By contrast, activation of the same plant defenses negatively affected feeding by the generalist herbivore S. exigua (van Oosten et al., 2008). Another factor that determines the effectiveness of root-colonizing microbe induced defenses is the insect feeding guilds. Chewing insects encounter several secondary metabolites when they feed on plant tissue. Piercing-sucking insects on the other hand avoid contact with these compounds by inserting their slender needle like stylets into the phloem sieve elements. Therefore, the degree of specialization and the feeding guild should be taken into account while considering the impact of beneficial soil-microbes on the aboveground herbivore. In order to recruit beneficial soil-dwelling organisms, plant roots produce copious amounts of exudates. The

290 V. J. Nalam et al. production of root exudates can therefore indirectly impact the outcome of plant interaction with the AGH. Root exudates comprise of an enormous range of small molecular weight compounds and the interactions mediated by these exudates can be either positive or negative (reviewed in Walker et al., 2003; Bais et al., 2006; Hiltpold et al., 2011). For instance, a large number of soil organisms rely on root exudates as a major source of carbon (Walker et al., 2003; Bais et al., 2006). On the other hand, root exudates also contain compounds with antimicrobial, nematicidal, or insecticidal properties. Several studies have shown that AGH influence soil microbial communities (Hamilton et al., 2008; van Dam, 2009). For example, silverleaf whitefly and green peach aphid feeding on pepper plants resulted in a significant increase in population density of several beneficial rhizobacteria (Yang et al., 2011; Lee et al., 2012). Whether root association with these rhizobacteria results in ISR against the phloem-feeding insects is unknown. It is however plausible to conclude from these and other studies (Murphy et al., 2000; Herman et al., 2008) that recruitment of beneficial rhizobacteria can have a negative impact on the AGH. These studies provide new insights into the tritrophic interactions between the AGH, the plant, and beneficial soil microbes. Herbivory induced root exudation can also result in unique soil legacy effects. For instance, AGH by M. brassicae on ragwort plants resulted in the production of root exudates that altered the composition of soil fungi. Plants grown subsequently in the same soil displayed increased biomass and higher content of pyrrolizidine alkaloids that enabled them to counter future threats by AGH (Kostenko et al., 2012). Roots are also involved in interplant communication via the interaction with the mycelia of mycorrhizal fungi. Mycorrhizal mycelia can interconnect roots of multiple plants to form common mycorrhizal netwroks (CMNs; Selosse et al., 2006). CMNs are known to act as conduits for the transfer of water and also nutrients such as nitrogen, phosphorous and other elements from one plant root to another (He et al., 2003; Meding & Zasoski, 2008; Mikkelsen et al., 2008). Exchange between plants connected by CMNs is not only limited to transfer of water and nutrients but also to the exchange of signals. For instance, CMNs mediate plant to plant communication between healthy and pathogen-infected tomato plants (Song et al., 2010). Pathogen-infected tomato plants transmit a defence signal to healthy plants where the expression of defence genes and activity is induced resulting in increased resistance to future attacks (Song et al., 2010). Although, a similar study with insects is lacking it is plausible to hypothesize that a similar exchange of defence signals between plants connected physically by CMNs also occurs during insect herbivory. The interaction of plant roots with mycorrhiza forming CMNs could therefore act as defence networks in plant communities. Root-colonizing microbes also contribute to indirect defenses against AGH by stimulating shoots to emit volatile organic compounds that attract natural enemies of AGH (Guerrieri et al., 2004). For instance, the rate of parasitism of the Bird cherry oat aphid, Rhopalosiphum padi,bythe parasitic wasp, Aphidius rhopalosiphi, increased by 140% on timothy grass (Phleum pratense) that were associated with arbuscular mycorrhizal fungi (Glomus intraradices) (Hempel et al., 2009). Additionally, belowground interaction of plants with root-colonizing microbes not only promotes plant growth but also has a beneficial effect on the nutritional status of the plant (Pineda et al., 2010). Root associations with soil-dwelling microbes enable the increased uptake of not only water but also a variety of nutrients such as nitrogen and phosphorus. Further, some microbes enhanced photosynthesis by modulating sugar and ABA signaling, and also synthesized plant growth promoting hormones (van Loon, 2007; Zhang et al., 2008). In general, increased content of nitrogen and other limiting nutrients in plant tissues and phloem sap benefits both chewing and phloem-sap feeding insects (Schoonhoven et al., 2005). The final result of root-colonizing microbes on AGH, therefore, depends on a fine balance between the positive effect on AGH due to enhanced nutritional status of the plant and negative effect on AGH due to promotion of induced resistance and indirect defenses in shoots. Signal(s) involved in shoot-to-root communication The involvement of roots in defense against AGH suggests communication by shoots with roots. Erb et al. (2009b) suggested the presence of a shoot root shoot loop in which signal(s) generated in response to AGH spread systemically throughout the plant including the roots. The translocation of the signal(s) to the roots results in the activation of root-based defenses, which then directly or indirectly influence the AGH. Several classes of compounds have the potential to act as signals. In order to qualify as a defense signal however, the compound(s) must be synthesized at the site of attack, from where it is systemically translocated to induce defense responses. Plant hormones such as JA and SA are critical signals in plant defense regulatory networks that are involved in long-distance signaling (Heil & Ton, 2008) and may therefore play a critical role in shoot-to-root communication. JA and associated compounds termed jasmonates are involved in long-distance wound signaling and are considered to be central to governing plant responses to chewing insects (Wu & Baldwin, 2010; Woldemariam

Root responses to aboveground herbivory 291 et al., 2011). Furthermore, methyl jasmonate is readily transported through the plants vasculature (Thorpe et al., 2007). Support for the role of jasmonates as shoot-toroot signals comes from studies on diverse plant insect interactions. In Nicotiana sylvestris, JA concentrations increased locally within 30 min of wounding and in the roots after 90 min resulting in the stimulation of nicotine synthesis (Baldwin et al., 1994; Winz & Baldwin, 2001). Foliar treatment of N. sylvestris with methyl jasmonate resulted in the upregulation of Putrescine N-methyltransferases, the enzymes that catalyze the first committed step of nicotine biosynthesis (Shoji et al., 2000). In poplar, simulated herbivory by the application of methyl jasmonate, or mechanical wounding, also resulted in the induction of a poplar trypsin inhibitor (PtdT13), a marker for poplar defenses, in both the leaves and roots (Major & Constabel, 2007). In Brassica rapa, foliar application of methyl jasmonate resulted in an increase in glucosinolate levels in the roots (Loivamäki et al., 2004). Furthermore, methyl jasmonate treatment resulted in the accumulation of Mir1- CP in maize leaves in a dose-dependent manner (Ankala et al., 2009). Since Mir1-CP is synthesized exclusively in the roots of maize plants, it is plausible that JA or its conjugates function as long-distance signals to stimulate Mir1-CP synthesis. Taken together, these results highlight the importance of jasmonates as important shoot-to-root signals during AGH by chewing insects. The role of SA and its derivative methyl salicylate in shoot-to-root communication is less clear. SA-mediated defenses play an important role in locally expressed defenses and in the enhancement of resistance to secondary infection in distal uninfected plant parts against biotrophic pathogens. The crosstalk between the SA and JA pathways is thought to modulate plant defense responses and limit the expression of costly and ineffective defenses (Glazebrook, 2005). Although, SA-based defenses are normally induced in response to pathogen attack, insect herbivory can also result in an increase in the levels of endogenous SA and/or the activation of SA-inducible genes (Moran & Thompson, 2001; Heidel & Baldwin, 2004; Zarate et al., 2007; Kanno et al., 2012). However, the lack of a negative effect on herbivore performance due to the activation of SA-mediated defenses (Moran & Thompson, 2001; Heidel & Baldwin, 2004; Zarate et al., 2007; Kanno et al., 2012) raises the possibility of herbivore-mediated manipulation of plant defenses. Indeed, nymphs of silverleaf whitefly induce SA-mediated defenses in order to suppress the more effective JA-mediated defenses (Zarate et al., 2007). However, the role of methyl salicylate as a signal molecule cannot be overlooked. Methyl salicylate in addition to being readily transported in the phloem is also volatile and is a key signaling molecule involved in plant-to-plant communication (Shulaev et al., 1997; Park et al., 2007). Furthermore, methyl salicylate mediates resistance against certain insects by attracting their respective predators (van Poecke & Dicke, 2002). Therefore, although SA and methyl salicylate may not be directly involved in shoot-to-root communication, they can indirectly influence the plants response to insect herbivory by modulating JA-mediated defenses. There may be yet undiscovered molecules that are involved in shoot-to-root communication. In maize plants infested with African cotton leafworm (S. littoralis), transcriptomic analyses revealed no overlap between the genes induced in the shoots and the roots. Furthermore, although JA and SA-mediated genes were induced in shoots in response to the AGH, the same set of genes were not induced in the roots during AGH suggesting the presence of alternative shoot-to-root signals (Erb dissertation, University of Neuchatel, 2009). In plants, small interfering RNA and micro RNA play an important role in plant defense responses to AGH (reviewed in Padmanabhan et al., 2009). For example, herbivory induced large-scale changes in the small RNA transcriptome of N. attenuata (Pandey et al., 2008). These small RNAs are thought to contribute to plant defenses by playing a central role in coordinating the large-scale transcriptional changes that occur in response to AGH. However, small RNAs are highly mobile and are readily transported in the phloem tissue and thus may have an important role in the regulation of systemic defenses (Yoo et al., 2004; Kehr & Buhtz, 2008). Further experimentation is however needed to confirm whether small RNAs play a role in shoot-to-root communication during AGH. The ability to simply and efficiently micrograft/graft various model species such as Arabidopsis, N. attenuata, Nicotiana benthamiana, and tomato (Voinnet et al., 2000; Turnbull et al., 2002; Kimura & Sinha, 2008; Fragoso et al., 2011), can greatly aid in evaluating the role of small RNAs and other candidates as potential shoot to root signals. In addition to plant-derived factors acting as shoot-to-root signals during AGH, insect elicitors may also function as signal molecules. Both chewing and piercing/sucking insects release a large repertoire of elicitors that are capable of inducing characteristic plant defense responses (Wu & Baldwin, 2009; Hogenhout & Bos, 2011). Insect elicitors have been identified in oral secretions, regurgitant, oviposition liquid, and saliva. Insect elicitors are not only capable of inducing JA, ethylene, and SA signaling but can also activate mitogen-activated protein kinases, produce reactive oxygen species and induce calcium ion fluxes (Wu & Baldwin, 2009). However, whether insect elicitors function as shoot-to-root signals directly or indirectly through plant-mediated mechanisms warrants further research.

292 V. J. Nalam et al. Conclusions Roots are play an integral role in plant defense against AGH by acting as sites of synthesis for various secondary metabolites and proteins that either kill or deter the herbivore from feeding. Roots also contribute to plant defenses indirectly by acting as temporary sites of storage for photoassimilates. Furthermore, the association of roots with beneficial soil-borne microorganisms not only aids in the growth and development of the plant, but also results in the induction of ISR and aids in the recruitment of natural enemies of insect herbivores. There is increasing evidence that plant signaling molecules such as JA and/or its derivatives, play an important role in shoot-to-root communication. There are several questions that are yet to be answered. For example, does the root response to AGH depend on the insect feeding guilds? Are the signal(s) involved in shoot-to-root communication derived from the plant and/or the insect? Do other phytohormones besides JA play a role in shoot-to-root communication? Does AGH alter root physiology to suppress host defenses? What is the metabolic cost to the plants due to root involvement in AGH defense? Answers to these and other relevant questions will provide a better understanding of the contribution of roots to plant defense against AGH. Acknowledgments We thank Dr. Keyan Zhu-Salzman for the invitation to contribute to this special issue. We would like to thank Dr. David C. Margolies for helpful comments on an earlier version of this manuscript. Work in the Shah lab was supported by grants from the National Science Foundation (Division of Integrative Organismal Systems-0919192 and Division of Molecular and Cellular Biosciences- 0920600). This work was supported by start-up funds from Indiana University Purdue University Fort Wayne to Dr. Punya Nachappa. Disclosure The views presented in the review article represent the author s views. The authors have declared no competing interests exist and are not involved in any potential conflicts of interest including financial interests, relationships and affiliations. References Aharoni, A., Jongsma, M.A., Kim, T.Y., Ri, M.B., Giri, A.P., Verstappen, F.W.A., Schwab, W. and Bouwmeester, H.J. (2006) Metabolic engineering of terpenoid biosynthesis in plants. Phytochemistry Reviews, 5, 49 58. Ankala, A., Luthe, D., Williams, W. and Wilkinson, J. (2009) Integration of ethylene and jasmonic acid signaling pathways in the expression of maize defense protein Mir1-CP. Molecular Plant Microbe Interactions, 22, 1555 1564. Babst, B.A., Ferrieri, R.A., Gray, D.W., Lerdau, M., Schlyer, D.J., Schueller, M., Thorpe, M.R. and Orians, C.M. (2005) Jasmonic acid induces rapid changes in carbon transport and partitioning in Populus. New Phytologist, 167, 63 72. Babst, B.A., Ferrieri, R.A., Thorpe, M.R. and Orians, C.M. (2008) Lymantria dispar herbivory induces rapid changes in carbon transport and partitioning in Populus nigra. Entomologia Experimentalis et Applicata, 128, 117 125. Bais, H.P., Weir, T.L., Perry, L.G., Gilroy, S. and Vivanco, J.M. (2006) The role of root exudates in rhizosphere interactions with plants and other organisms. Annual Review of Plant Biology, 57, 233 266. Baldwin, I.T., Schmelz, E.A. and Ohnmeiss, T.E. (1994) Woundinduced changes in root and shoot jasmonic acid pools correlate with induced nicotine synthesis in Nicotiana sylvestris Spegazzini and Comes. Journal of Chemical Ecology, 20, 2139 2157. Berenbaum, M. (1978) Toxicity of a furanocoumarin to armyworms: A case of biosynthetic escape from insect herbivores. Science, 201, 532. Bezemer, T., Wagenaar, R., van Dam, N.M., van der Putten, W. and Wäckers, F. (2004) Above-and below-ground terpenoid aldehyde induction in cotton, Gossypium herbaceum, following root and leaf injury. Journal of Chemical Ecology, 30, 53 67. Bezemer, T.M. and van Dam, N.M. (2005) Linking aboveground and belowground interactions via induced plant defenses. Trends in Ecology & Evolution, 20, 617 624. Bezemer, T.M., Wagenaar, R., van Dam, N.M. and Wäckers, F.L. (2003) Interactions between above- and belowground insect herbivores as mediated by the plant defense system. Oikos, 101, 555 562. Blossey, B. and Hunt-Joshi, T.R. (2003) Belowground herbivory by insects: Influence on plants and aboveground herbivores. Annual Review of Entomology, 48, 521 547. Bostock, R.M. (2005) Signal crosstalk and induced resistance: Straddling the line between cost and benefit. Annual Review of Phytopathology, 43, 545 580. Chen, M.S. (2008) Inducible direct plant defense against insect herbivores: A review. Insect Science, 15, 101 114. Chen, S., Petersen, B.L., Olsen, C.E., Schulz, A. and Halkier, B.A. (2001) Long-distance phloem transport of glucosinolates in Arabidopsis. Plant Physiology, 127, 194 201. Cherqui, A. and Tjallingii, W.F. (2000) Salivary proteins of aphids, a pilot study on identification, separation and

Root responses to aboveground herbivory 293 immunolocalisation. Journal of Insect Physiology, 46, 1177 1186. Cipollini, D., Purrington, C. and Bergelson, J. (2003) Costs of induced responses. Basic and Applied Ecology, 28, 161 174. Collantes, H.G., Gianoli, E. and Niemeyer, H.M. (1999) Defoliation affects chemical defenses in all plant parts of rye seedlings. Journal of Chemical Ecology, 25, 491 499. Dawson, R.F. (1941) The localization of the nicotine synthetic mechanism in the tobacco plant. Science, 94, 396 397. Dempsey, D. and Klessig, D.F. (2012) SOS too many signals for systemic acquired resistance? Trends in Plant Science, 17, 538 545. Doornbos, R.F., van Loon, L.C. and Bakker, P.A.H.M. (2010) Impact of root exudates and plant defense signaling on bacterial communities in the rhizosphere. A review. Agronomy for Sustainable Development, 32, 227 243. Erb, M., Flors, V., Karlen, D., De Lange, E., Planchamp, C., D alessandro, M., Turlings, T.C.J. and Ton, J. (2009a) Signal signature of aboveground-induced resistance upon belowground herbivory in maize. Plant Journal, 59, 292 302. Erb, M., Lenk, C., Degenhardt, J. and Turlings, T.C.J. (2009b) The underestimated role of roots in defense against leaf attackers. Trends in Plant Science, 14, 653 659. Erb, M. (2012) The role of roots in plant defence. Plant Defence: Biological Control (eds. J.M.M. Mérillon & K.G.G. Ramawats), pp. 291 309. Springer, Netherlands. Erb, M., Glauser, G. and Robert, C.a.M. (2012) Induced immunity against belowground insect herbivores-activation of defenses in the absence of a jasmonate burst. Journal of Chemical Ecology, 1 12. Facchini, P.J. (2001) Alkaloid biosynthesis in plants: Biochemistry, cell biology, molecular regulation, and metabolic engineering applications. Annual Review of Plant Biology, 52, 29 66. Fragoso, V., Goddard, H., Baldwin, I.T. and Kim, S.G. (2011) A simple and efficient micrografting method for stably transformed Nicotiana attenuata plants to examine shoot-root signaling. Plant Methods,7,34. Glazebrook, J. (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annual Review of Phytopathology, 43, 205 227. Gómez, S., Steinbrenner, A.D., Osorio, S., Schueller, M., Ferrieri, R.A., Fernie, A.R. and Orians, C.M. (2012) From shoots to roots: Transport and metabolic changes in tomato after simulated feeding by a specialist lepidopteran. Entomologia Experimentalis et Applicata, 144, 101 111. Guerrieri, E., Lingua, G., Digilio, M.C., Massa, N. and Berta, G. (2004) Do interactions between plant roots and the rhizosphere affect parasitoid behaviour? Ecological Entomology, 29, 753 756. Halford, N. and Hey, S. (2009) Snf1-related protein kinases (SnRKs) act within an intricate network that links metabolic and stress signalling in plants. Biochemical Journal, 419, 247 259. Hamilton, E.W., Frank, D.A., Hinchey, P.M. and Murray, T.R. (2008) Defoliation induces root exudation and triggers positive rhizospheric feedbacks in a temperate grassland. Soil Biology and Biochemistry, 40, 2865 2873. Hartmann, T. and Ober, D. (2000) Biosynthesis and metabolism of pyrrolizidine alkaloids in plants and specialized insect herbivores. Biosynthesis, 207 243. He, X.H., Critchley, C. and Bledsoe, C. (2003) Nitrogen transfer within and between plants through common mycorrhizal networks (CMNs). Critical Reviews in Plant Sciences, 22, 531 567. Heidel, A. and Baldwin, I.T. (2004) Microarray analysis of salicylic acid- and jasmonic acid-signalling in responses of Nicotiana attenuata to attack by insects from multiple feeding guilds. Plant, Cell & Environment, 27, 1362 1373. Heil, M. and Ton, J. (2008) Long-distance signalling in plant defence. Trends in Plant Science, 13, 264 272. Hempel, S., Stein, C., Unsicker, S.B., Renker, C., Auge, H., Weisser, W.W. and Buscot, F. (2009) Specific bottom up effects of arbuscular mycorrhizal fungi across a plant herbivore parasitoid system. Oecologia, 160, 267 277. Herman, M., Nault, B. and Smart, C. (2008) Effects of plant growth-promoting rhizobacteria on bell pepper production and green peach aphid infestations in New York. Crop Protection, 27, 996 1002. Hiltpold, I., Erb, M., Robert, C.a.M. and Turlings, T.C.J. (2011) Systemic root signalling in a belowground, volatile-mediated tritrophic interaction. Plant Cell and Environment, 34, 1267 1275. Hogenhout, S.A. and Bos, J.I.B. (2011) Effector proteins that modulate plant-insect interactions. Current Opinion in Plant Biology, 14, 422 428. Hol, G., Macel, M., van Veen, J.A. and van der Meijden, E. (2004) Root damage and aboveground herbivory change concentration and composition of pyrrolizidine alkaloids of Senecio jacobaea. Basic and Applied Ecology, 5, 253 260. Holland, J.N., Cheng, W. and Crossley, D. (1996) Herbivoreinduced changes in plant carbon allocation: Assessment of below-ground C fluxes using carbon-14. Oecologia, 107, 87 94. Howe, G.A. and Jander, G. (2008) Plant immunity to insect herbivores. Annual Review of Plant Biology, 59, 41 66. Johnson, S., Bezemer, T. and Jones, T. (2008) Linking aboveground and belowground herbivory. Root Feeders: An Ecosystem Persepctive (eds. S.N. Johnson & P.J. Murray), pp. 153 170. CABI, Wallingford, UK.

294 V. J. Nalam et al. Kanno, H., Hasegawa, M. and Kodama, O. (2012) Accumulation of salicylic acid, jasmonic acid and phytoalexins in rice, Oryza sativa, infested by the white-backed planthopper, Sogatella furcifera (Hemiptera: Delphacidae). Applied Entomology and Zoology, 47, 27 34. Kaplan, I., Halitschke, R., Kessler, A., Rehill, B.J., Sardanelli, S. and Denno, R.F. (2008a) Physiological integration of roots and shoots in plant defense strategies links above- and belowground herbivory. Ecology Letters, 11, 841 851. Kaplan, I., Halitschke, R., Kessler, A., Sardanelli, S. and Denno, R.F. (2008b) Constitutive and Induced defenses to herbivory in above- and belowground plant tissues. Ecology, 89, 392 406. Karban, R. and Baldwin, I.T. (1997) Induced Responses to Herbivory. Chicago University Press, Chicago. Kehr, J. and Buhtz, A. (2008) Long distance transport and movement of RNA through the phloem. Journal of Experimental Botany, 59, 85 92. Kimura, S. and Sinha, N. (2008) Grafting tomato plants. Cold Spring Harbor Protocols 2008, pdb. prot5083. Kostenko, O., van de Voorde, T.F.J., Mulder, P.P.J., van der Putten, W.H. and Bezemer, T.M. (2012) Legacy effects of aboveground belowground interactions. Ecology Letters, 15, 813 821. Kempel, A., Brandl, R. and Schädler, M. (2009) Symbiotic soil microorganisms as players in aboveground plant herbivore interactions the role of rhizobia. Oikos, 118, 634 640. Lee, B., Lee, S. and Ryu, C.M. (2012) Foliar aphid feeding recruits rhizosphere bacteria and primes plant immunity against pathogenic and non-pathogenic bacteria in pepper. Annals of Botany, 110, 281 290. Loivamäki, M., Holopainen, J.K. and Nerg, A.M. (2004) Chemical changes induced by methyl jasmonate in oilseed rape grown in the laboratory and in the field. Journal of Agricultural and Food Chemistry, 52, 7607 7613. Lopez, L., Camas, A., Shivaji, R., Ankala, A., Williams, P. and Luthe, D. (2007) Mir1-CP, a novel defense cysteine protease accumulates in maize vascular tissues in response to herbivory. Planta, 226, 517 527. Ludwig-Müller, J., Schubert, B., Pieper, K., Ihmig, S. and Hilgenberg, W. (1997) Glucosinolate content in susceptible and resistant Chinese cabbage varieties during development of clubroot disease. Phytochemistry, 44, 407 414. Major, I.T. and Constabel, C.P. (2007) Shoot-root defense signaling and activation of root defense by leaf damage in poplar. Botany, 85, 1171 1181. Meding, S. and Zasoski, R. (2008) Hyphal-mediated transfer of nitrate, arsenic, cesium, rubidium, and strontium between arbuscular mycorrhizal forbs and grasses from a California oak woodland. Soil Biology and Biochemistry, 40, 126 134. Mikkelsen, B.L., Rosendahl, S. and Jakobsen, I. (2008) Underground resource allocation between individual networks of mycorrhizal fungi. New Phytologist, 180, 890 898. Moran, P.J. and Thompson, G.A. (2001) Molecular responses to aphid feeding in Arabidopsis in relation to plant defense pathways. Plant Physiology, 125, 1074 1085. Morita, M., Shitan, N., Sawada, K., van Montagu, M.C.E., Inzé, D., Rischer, H., Goossens, A., Oksman-Caldentey, K.M., Moriyama, Y. and Yazaki, K. (2009) Vacuolar transport of nicotine is mediated by a multidrug and toxic compound extrusion (MATE) transporter in Nicotiana tabacum. Proceedings of the National Academy of Sciences of the United States of America, 106, 2447 2452. Murphy, J.F., Zehnder, G.W., Schuster, D.J., Sikora, E.J., Polston, J.E. and Kloepper, J.W. (2000) Plant growth-promoting rhizobacterial mediated protection in tomato against Tomato mottle virus. Plant Disease, 84, 779 784. Nalam, V.J., Keeretaweep, J., Sarowar, S. and Shah, J. (2012) Root-derived oxylipins promote green peach aphid performance on Arabidopsis foliage. The Plant Cell, 24, 1643 1653. Nandi, B., Kundu, K., Banerjee, N. and Babu, S.P.S. (2003) Salicylic acid-induced suppression of Meloidogyne incognita infestation of okra and cowpea. Nematology, 5, 747 752. Öpik, H., Rolfe, S.A., Willis, A.J. and Street, H.E. (2005) The Physiology of Flowering Plants. Cambridge University Press. Orians, C.M., Thorn, A. and Gómez, S. (2011) Herbivoreinduced resource sequestration in plants: Why bother? Oecologia, 167, 1 9. Padmanabhan, C., Zhang, X. and Jin, H. (2009) Host small RNAs are big contributors to plant innate immunity. Current Opinion in Plant Biology, 12, 465 472. Pandey, S.P., Shahi, P., Gase, K. and Baldwin, I.T. (2008) Herbivory-induced changes in the small-rna transcriptome and phytohormone signaling in Nicotiana attenuata. Proceedings of the National Academy of Sciences of the United States of America, 105, 4559. Park, S.W., Kaimoyo, E., Kumar, D., Mosher, S. and Klessig, D.F. (2007) Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science, 318, 113. Pineda, A., Zheng, S.J., van Loon, J.J.A., Pieterse, C.M.J. and Dicke, M. (2010) Helping plants to deal with insects: The role of beneficial soil-borne microbes. Trends in Plant Science, 15, 507 514. Raps, A. and Vidal, S. (1998) Indirect effects of an unspecialized endophytic fungus on specialized plant herbivorous insect interactions. Oecologia, 114, 541 547. Rasmann, S. and Agrawal, A.A. (2008) In defense of roots: A research agenda for studying plant resistance to belowground herbivory. Plant Physiology, 146, 875 880. Roitsch, T. and González, M.C. (2004) Function and regulation of plant invertases: Sweet sensations. Trends in Plant Science, 9, 606 613.

Root responses to aboveground herbivory 295 Saravanakumar, D., Muthumeena, K., Lavanya, N., Suresh, S., Rajendran, L., Raguchander, T. and Samiyappan, R. (2007) Pseudomonas-induced defence molecules in rice plants against leaffolder (Cnaphalocrocis medinalis) pest. Pest Management Science, 63, 714 721 Schoonhoven, L.M., van Loon, J.J.A. and Dicke, M. (2005) Insect Plant Biology. Oxford University Press. Schwachtje, J., Minchin, P.E.H., Jahnke, S., van Dongen, J.T., Schittko, U. and Baldwin, I.T. (2006) SNF1-related kinases allow plants to tolerate herbivory by allocating carbon to roots. Proceedings of the National Academy of Sciences of the United States of America, 103, 12935 12940. Selosse, M.A., Richard, F., He, X. and Simard, S.W. (2006) Mycorrhizal networks: Des liaisons dangereuses? Trends in Ecology & Evolution, 21, 621 628. Shah, J. (2009) Plants under attack: Systemic signals in defence. Current Opinion in Plant Biology, 12, 459 464. Shoji, T., Yamada, Y. and Hashimoto, T. (2000) Jasmonate induction of putrescine N-methyltransferase genes in the root of Nicotiana sylvestris. Plant and Cell Physiology, 41, 831 839. Shulaev, V., Silverman, P. and Raskin, I. (1997) Airborne signalling by methyl salicylate in plant pathogen resistance. Nature, 385, 718 721. Sidwa-Gorycka, M., Królicka, A., Kozyra, M., Głowniak, K., Bourgaud, F. and Łojkowska, E. (2003) Establishment of a co-culture of Ammi majus L. and Ruta graveolens L. for the synthesis of furanocoumarins. Plant Science, 165, 1315 1319. Smith, C.M. (2005) Plant Resistance to Arthropods: Molecular and Conventional Approaches, Kluwer Academic Pub. Soler, R., Schaper, S.V., Bezemer, T., Cortesero, A.M., Hoffmeister, T.S., van der Putten, W.I.M.H., Vet, L.E.M. and Harvey, J.A. (2009) Influence of presence and spatial arrangement of belowground insects on host-plant selection of aboveground insects: A field study. Ecological Entomology, 34, 339 345. Song, Y.Y., Zeng, R.S., Xu, J.F., Li, J., Shen, X. and Yihdego, W.G. (2010) Interplant communication of tomato plants through underground common mycorrhizal networks. PloS ONE, 5, e13324. Steinbrenner, A.D., Gómez, S., Osorio, S., Fernie, A.R. and Orians, C.M. (2011) Herbivore-induced changes in tomato (Solanum lycopersicum) primary metabolism: A whole plant perspective. Journal of Chemical Ecology, 37, 1294 1303. Thorpe, M.R., Ferrieri, A.P., Herth, M.M. and Ferrieri, R.A. (2007) 11 C-imaging: Methyl jasmonate moves in both phloem and xylem, promotes transport of jasmonate, and of photoassimilate even after proton transport is decoupled. Planta, 226, 541 551. Toppel, G., Witte, L., Riebesehl, B., Borstel, K. and Hartmann, T. (1987) Alkaloid patterns and biosynthetic capacity of root cultures from some pyrrolizidine alkaloid producing Senecio species. Plant Cell Reports, 6, 466 469. Turgeon, R. and Wolf, S. (2009) Phloem transport: Cellular pathways and molecular trafficking. Annual Review of Plant Biology, 60, 207 221. Turnbull, C.G.N., Booker, J.P. and Leyser, H.M.O. (2002) Micrografting techniques for testing long-distance signalling in Arabidopsis. Plant Journal, 32, 255 262. Valenzuela-Soto, J.H., Estrada-Hernández, M.G., Ibarra- Laclette, E. and Délano-Frier, J.P. (2010) Inoculation of tomato plants (Solanum lycopersicum) with growthpromoting Bacillus subtilis retards whitefly Bemisia tabaci development. Planta, 231, 397 410. van Dam, N.M. and Vrieling, K. (1994) Genetic variation in constitutive and inducible pyrrolizidine alkaloid levels in Cynoglossum officinale L. Oecologia, 99, 374 378. van Dam, N.M., Horn, M., Mareš, M. and Baldwin, I.T. (2001) Ontogeny constrains systemic protease inhibitor response in Nicotiana attenuata. Journal of Chemical Ecology, 27, 547 568. van Dam, N.M. (2009) Belowground herbivory and plant defenses. Annual Review of Ecology Evolution and Systematics, 40, 373 391. van der Putten, W.H., Vet, L.E.M., Harvey, J.A. and Wäckers, F.L. (2001) Linking above- and belowground multitrophic interactions of plants, herbivores, pathogens, and their antagonists. Trends in Ecology & Evolution, 16, 547 554. van Loon, L. (2007) Plant responses to plant growth-promoting rhizobacteria. European Journal of Plant Pathology, 119, 243 254. van Oosten, V.R., Bodenhausen, N., Reymond, P., van Pelt, J.A., van Loon, L.C., Dicke, M. and Pieterse, C.M.J. (2008) Differential effectiveness of microbially induced resistance against herbivorous insects in Arabidopsis. Molecular Plant Microbe Interactions, 21, 919 930. van Poecke, R.M.P. and Dicke, M. (2002) Induced parasitoid attraction by Arabidopsis thaliana: Involvement of the octadecanoid and the salicylic acid pathway. Journal of Experimental Botany, 53, 1793 1799. Vlot, A.C., Dempsey, D.M.A. and Klessig, D.F. (2009) Salicylic acid, a multifaceted hormone to combat disease. Annual Review of Phytopathology, 47, 177 206. Vlot, A.C., Klessig, D.F. and Park, S.W. (2008) Systemic acquired resistance: The elusive signal(s). Current Opinion in Plant Biology, 11, 436 442. Voinnet, O., Lederer, C. and Baulcombe, D.C. (2000) A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell, 103, 157 167. Walker, T.S., Bais, H.P., Grotewold, E. and Vivanco, J.M. (2003) Root exudation and rhizosphere biology. Plant Physiology, 132, 44 51.