Herbivore attack leads to resource conflicts between plant defensive strategies. Photoassimilates are required for defensive compounds and carbon storage below ground and may therefore be depleted or enriched in the roots of herbivore-defoliated plants. The potential role of belowground tissues as mediators of induced tolerance–defense trade-offs is unknown.
We evaluated signaling and carbohydrate dynamics in the roots of Nicotiana attenuata following Manduca sexta attack. Experimental and natural genetic variability was exploited to link the observed metabolite patterns to plant tolerance and resistance.
Leaf-herbivore attack decreased sugar and starch concentrations in the roots and reduced regrowth from the rootstock and flower production in the glasshouse and the field. Leaf-derived jasmonates were identified as major regulators of this root-mediated resource-based trade-off: lower jasmonate levels were associated with decreased defense, increased carbohydrate levels and improved regrowth from the rootstock. Application and transport inhibition experiments, in combination with silencing of the sucrose non-fermenting (SNF) -related kinase GAL83, indicated that auxins may act as additional signals that regulate regrowth patterns.
In conclusion, our study shows that the ability to mobilize defenses has a hidden resource-based cost below ground that constrains defoliation tolerance. Jasmonate- and auxin-dependent mechanisms may lead to divergent defensive plant strategies against herbivores in nature.
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Plants have evolved mechanisms that allow them to maximize protection against herbivorous insects whilst minimizing deviations from optimal growth and fitness (Meldau et al., 2012). Defensive metabolites and proteins, for instance, have been found to accumulate on herbivore attack (Green & Ryan, 1972; Baldwin et al., 1998; Zhu-Salzman et al., 2008; Glauser et al., 2011). However, herbivores can adapt to toxic and deterrent compounds (Zangerl & Berenbaum, 1990; Berenbaum & Zangerl, 1994; Lindigkeit et al., 1997; Mao et al., 2006). Possibly as a consequence, plants have evolved alternative strategies that may reduce herbivore-imposed fitness costs even in the face of resistant attackers. The induction of meristematic activity, photosynthesis and reallocation of resources, for instance, may increase plant tolerance and help attacked individuals to grow and produce offspring even under heavy herbivore pressure (reviewed by Strauss & Agrawal, 1999; Tiffin, 2000; Schwachtje & Baldwin, 2008). Recent evidence suggests that many plants employ mixed tolerance–resistance strategies (Leimu & Koricheva, 2006; Núñez-Farfán et al., 2007; Carmona & Fornoni, 2013), allowing them to cope with the spatially and temporally diverse herbivore communities which they face in nature.
A growing body of evidence suggests that plants respond to actual and simulated herbivory by increasing the remobilization of resources from damaged and undamaged tissues to stems and roots, a process termed ‘herbivory-induced resource sequestration’ (Dyer et al., 1991; Briske et al., 1996; Holland et al., 1996; Babst et al., 2005, 2008; Bazot et al., 2005; Schwachtje et al., 2006; Kaplan et al., 2008; Gómez et al., 2010, 2012). The molecular basis of this phenomenon has been studied in Nicotiana attenuata. Schwachtje et al. (2006) found that the β-subunit of the sucrose non-fermenting-related kinase (SnRK1), GAL83, is down-regulated in source leaves within hours following simulated attack by Manduca sexta. GAL83-silenced plants constitutively allocated 10% more photoassimilates to the roots and had a prolonged flowering period on water deprivation (Schwachtje et al., 2006). Although these results speak in favor of the hypothesis that induced resource sequestration may be part of a plant's tolerance response, other studies report that attacked plants import more resources into the leaves to support plant defenses (Arnold & Schultz, 2002; Arnold et al., 2004; Appel et al., 2012; Ferrieri et al., 2012, 2013). Indeed, resource reallocation does not necessarily result in increased carbohydrate pools (Schwachtje et al., 2006), as the translocated assimilates may be used for other processes, such as exudation into the rhizosphere (Holland et al., 1996; Frost & Hunter, 2008), respiration (Clayton et al., 2010) and the synthesis of defensive metabolites (Shoji et al., 2000). It is noteworthy in this context that no increase in root primary metabolite pools following leaf attack has been demonstrated so far in any of the above plant systems (see, for example, Schwachtje et al., 2006). On the contrary, a recent study in tomato has shown that M. sexta attack triggers carbon (C) reallocation to the roots and, at the same time, leads to a depletion of carbohydrates (Gómez et al., 2012). Clearly, the connection between resource reallocation and tolerance deserves more attention if we are to understand the role of primary metabolism in plant defensive strategies.
Jasmonates (JA) are important regulators of plant resistance (reviewed by Liechti & Farmer, 2002) and have been implicated in systemic signaling between leaves and roots (Zhang & Baldwin, 1997). On herbivore attack, JA levels increase in local and systemic tissues and trigger the biosynthesis of many defensive metabolites (Howe & Jander, 2008), including, for instance, nicotine in N. attenuata (Steppuhn et al., 2004). JA deficiency results in a strong decrease in induced defenses and renders plants susceptible to a wide range of herbivores (Paschold et al., 2007; Kallenbach et al., 2012). At the same time, JA reduce plant growth and defoliation tolerance, suggesting that they mediate herbivore-induced trade-offs between resistance and tolerance (Zavala et al., 2006). Two explanations have been proposed for this phenomenon. First, JA may antagonize gibberellin (GA), cytokinin and auxin signaling, which may reduce cell elongation and growth directly (Chen et al., 2011; Yang et al., 2012). Second, JA-dependent defenses may deplete plant resources and thereby limit plant growth. The role of JA in tolerance–resistance trade-offs is complicated by the finding that the exogenous application of methyl jasmonate (MeJA) induces C reallocation to the roots of poplar and Arabidopsis thaliana (Babst et al., 2005, 2008; Ferrieri et al., 2013) and nitrogen in tomato (Gómez et al., 2010), whereas changes in C reallocation were found to be independent of JA in N. attenuata (Schwachtje et al., 2006). As JA mediate systemic signals that reprogram both primary and secondary metabolism in the leaves and the roots, detailed mechanistic studies will be crucial to evaluate the role of JA signaling in defense and tolerance.
We aimed to obtain an understanding of the role of plant roots in tolerance–defense trade-offs in N. attenuata. This plant responds to herbivory by the synthesis of alkaloids in the roots which are then transported to the leaves for defense (Baldwin et al., 1997). At the same time, root allocation of photoassimilates increases (Schwachtje et al., 2006), making the species a suitable choice to investigate the role of roots in tolerance and resistance. In the current study, we specifically focused on whether the enrichment or depletion of photoassimilates in the roots following leaf-herbivore attack alters the regrowth capacity of N. attenuata from the roots. To this end, we used genetically engineered N. attenuata lines and a set of diverse field-collected genotypes which vary in their defensive strategies. To understand the metabolic processes that govern tolerance–defense trade-offs, we measured phytohormone, defensive metabolite and major carbohydrate levels in the leaves and roots of N. attenuata. Our results provide a comprehensive picture of the impact of leaf induction on the metabolism and regrowth capacity of plants as a function of herbivore-induced phytohormone signaling across experimental and natural genetic variation, and suggest that roots play an important and possibly underestimated role in tolerance–defense trade-offs.
Materials and Methods
Plant material and planting conditions
The following plant material was used in the present study: wild-type N. attenuata Torr. Ex. Watson plants of the 31st inbred generation derived from seeds collected at the Desert Inn Ranch in Utah, UT, USA in 1988; an anti-sense line silenced in the expression of the sucrose non-fermenting 1 (SNF1)-related kinase GAL83 (asGAL83; Schwachtje et al., 2006); a JA-deficient inverted repeat allene oxide cyclase line (irAOC; Kallenbach et al., 2012); a transgenic control line transformed with an empty vector construct (EV, line A-03-9-1); a set of 120 ecotypes grown from seeds that were collected over 20 yr from different locations in the Great Basin Desert (UT, USA). Before planting, all seeds were surface sterilized and germinated on Gamborg's B5 medium, as described by Krügel et al. (2002). For glasshouse experiments, the seedlings were transferred to Teku pots (Pöppelmann GmbH & Co. KG, Lohne, Germany) 10 d after germination and, 10–12 d later, the seedlings were planted into 1-l pots filled with washed sand. Plants were grown as described by Krügel et al. (2002). For the field experiment, seeds of the transformed N. attenuata lines were imported under APHIS notification number 07-341-101n and experiments were conducted under notification number 06-242-02r. Plants were grown as described by Schuman et al. (2012).
Regrowth capacity from the roots following M. sexta attack
To understand how Manduca sexta (Linnaeus) attack affects the regrowth capacity of N. attenuata from the roots, we placed six neonate larvae on wild-type plants and let them feed freely for 6 d (n =30). Non-infested plants (n =30) were used as controls. To specifically investigate the role of the roots in supplying resources for leaf growth, all stems and leaves were removed (hereafter referred to as ‘shoot removal’), together with the larvae, 6 d after infestation, leaving only the root system and the lowest 0.5 cm of the stalk for regrowth (hereafter referred to as ‘remaining shoot’). Complete shoot removal by browsing mammals can occur under natural conditions (Baldwin, 1998). The regrowth and fitness of the regrowing plants were monitored by determining the average rosette diameter, branch length and number of flowers. All these parameters have been found to be positively correlated with total seed production (Glawe et al., 2003). To determine whether the presence of the main stem affects the regrowth capacity following M. sexta attack, we induced plants by simulated herbivory (W + OS) and either removed the complete shoot or only the leaves. Regrowth was then measured as described above (n =5). Herbivory was simulated by wounding (W) three leaves with a pattern wheel to produce three rows on each side of the midvein and treating the wounds immediately with 10 μl of a 1 : 5-diluted M. sexta oral secretions (OS) solution. This treatment mimics the defense induction triggered by M. sexta without removing extensive amounts of leaf-tissue (Qu et al., 2004). Every 48 h, three leaves per plant were treated over a total of 6 d, resulting in nine treated leaves per plant.
Regrowth capacity from the roots in the field
To evaluate the consequences of leaf induction for the regrowth capacity of N. attenuata under natural conditions, we conducted a field experiment in the Lytle Ranch Preserve (UT, USA). EV and JA-deficient irAOC plants were planted on 14 May 2012 in quadruplets. Once the plants reached the rosette stage, one EV and one irAOC plant per quadruplet were subjected to simulated herbivory (W + OS) as described above (n =14). Control plants were left untreated. One day after the last treatment, the shoots were removed as described above. Nine days after shoot removal, the rosette diameters of the regrowing shoots were measured. Plants that died during the regrowing phase were removed from the analysis (EV W + OS, 2; EV control, 2; irAOC W + OS, 5; irAOC control, 4).
Resource mobilization for regrowth
To investigate whether N. attenuata roots can mobilize resources to regrow shoots in the absence of residual photosynthetic activity, we induced plants as described above and removed their shoots 6 d after the first treatment. Half of the plants were harvested immediately after shoot removal, and their root and remaining shoot biomass was determined (n =15). The other half was left to regrow in total darkness for 9 d, after which their root, remaining shoot and regrown leaf biomasses were determined (n =15). One plant was excluded from the analysis as a result of pathogen contamination of the rootstock.
Effect of induction on regrowth capacity under water stress
Under natural conditions, soil desiccation is thought to function as an abiotic signal that is used by N. attenuata to mobilize its remaining root resources for a final reproductive effort at the end of the growing season (Schwachtje et al., 2006). However, water stress also induces C reallocation to the roots (Geiger & Servaites, 1991). To determine whether soil moisture characteristics affect the regrowth capacity of N. attenuata from the roots following leaf induction, we treated EV and JA-deficient irAOC plants with wounding and M. sexta oral secretions (W + OS) or wounding and water (W + W) as described above, and gradually reduced the water supply during the 6 d of induction treatments. For this, pots of control plants were weighed daily, and the weight loss caused by evaporation and transpiration was compensated by watering. Water-stressed plants only received a fraction of the water they had lost: over the 6 d of water stress, the amount of resupplied water was reduced from 90% to 50%. After shoot removal, all the plants were again watered normally (n =12).
Regrowth capacity of JA-deficient and allocation-altered transgenic plants
An additional experiment was conducted to evaluate the contribution of JA signaling and the SNF1-related kinase GAL83 to the regrowth capacity of N. attenuata in detail. asGAL83 plants have been shown to constitutively allocate more carbohydrates to the roots (Schwachtje et al., 2006). Thus, we hypothesized that these plants would have an increased capacity to regrow from the rootstock. Plants transformed with an empty vector (EV) were used as controls (n =12). Plants were induced by wounding and applications of M. sexta oral secretions or water as described above. Control plants were left intact. In addition, irAOC plants were complemented with MeJA to restore JA signaling. For this, 75μg of MeJA in lanolin paste were applied to one leaf every other day for 6 d. Following the different treatments, the shoots of all plants were removed and the regrowth capacity was monitored as described above.
The role of leaf- and root-derived JA as regulators of regrowth
Root- and leaf-derived JA are likely to have different functions in plant stress responses, growth and development (reviewed by Wasternack & Hause, 2013). To determine the role of leaf- and root-derived JA on the regrowth responses of N. attenuata, we monitored the regrowth capacity after leaf herbivory of different JA-deficient chimeric plants. The chimeric plants were created by micrografting according to the procedures described in Fragoso et al. (2011). The following micrografting combinations were evaluated: EV/EV, plants with intact JA signaling (n =10); EV/irAOC, plants silenced in root JA production (n =8); irAOC/irAOC, plants silenced in both root and leaf JA production (n =10). The different grafting combinations were treated and monitored for regrowth as described above.
Herbivory-induced reconfiguration of primary and secondary metabolism in leaves and roots
To determine whether leaf herbivory reconfigures leaf and root primary and secondary metabolism, we treated leaves of EV, asGAL83 and irAOC plants as described above, harvested the shoots and roots 6 d after the first treatment and 6 d after the start of regrowth, and measured nicotine, soluble sugars and starch in the different tissues (n =3). To determine the concentration of nicotine, previously described procedures were followed (Keinänen et al., 2001). Soluble sugars were extracted from plant tissue using 80% (v/v) ethanol, followed by an incubation step (10 min at 78°C) with constant shaking at 800 rpm. Pellets were re-extracted twice with 50% (v/v) ethanol (10 min at 78°C with constant shaking at 800 rpm). Supernatants from all extraction steps were pooled together, and sucrose, glucose and fructose were quantified as described by Velterop & Vos (2001). The remaining pellets were used for an enzymatic determination of starch content as described previously (Smith & Zeeman, 2006).
To find possible systemic signals that trigger the herbivory-induced reduction in regrowth from the rootstock, we evaluated the induction of phytohormones in response to M. sexta attack. For this, three rosette leaves were wounded and immediately treated with 30 μl of a 1 : 5 (v/v) milliQ water-diluted M. sexta oral secretions solution (W + OS). Wounded and water-treated plants (W + W), as well as intact plants, were used as controls. Roots and leaves were harvested 30 min, 1 and 3 h after treatment. The extraction and quantification of phytohormones were carried out as described by Glauser et al. (2012) with some modifications (see Supporting Information Note S1 for details).
JA signaling and regrowth in natural populations
To examine potential trade-offs between JA signaling and the regrowth capacity in natural populations of N. attenuata, we analyzed the regrowth capacity and herbivory-induced JA production of 120 different individuals originating from different natural accessions. To quantify the herbivore-induced jasmonic acid production, the S1 leaf was wounded by rolling a fabric pattern wheel three times on one side of the midvein. The wounds were immediately treated with 10 μl of a 1 : 5 (v/v) milliQ water-diluted M. sexta oral secretions solution. The S1 leaf is the youngest fully developed leaf and highly responsive to insect attack (Zavala & Baldwin, 2004). After 60 min, the tissue of the treated leaf side was collected and immediately frozen in liquid nitrogen. JA was quantified as described previously (Stitz et al., 2011). To phenotype the regrowth capacity, the shoots of all plants were removed at the end of the flowering period and the regrowth capacity was measured as described above. This experiment was embedded in a large sampling campaign that measured a number of phenotypic traits in different N. attenuata accessions. Additional results from this experiment will be published elsewhere.
The role of GAs as regulators of regrowth
To determine the possible role of GAs in the observed regrowth effects, we induced plants as described above and monitored the internode elongation of regrowing branches. Internode elongation has been used as a downstream marker to determine JA-induced alterations in GA signaling (Yang et al., 2012). The average internode length was determined by measuring the length of the longest branch and counting its number of internodes. In addition, we determined the number of branches and total length of the branches (cumulative branching; n =10).
The role of auxins as regulators of regrowth
To determine the importance of auxin (indole-3-acetic acid, IAA) on the regulation of root responses to insect attack, we applied either 0.7 mg IAA or 0.3 mg trans-cinnamic acid (TCA) dissolved in lanolin paste to the petioles of N. attenuata immediately after W + OS elicitation. TCA has been shown to alter auxin polar transport (An et al., 1999). W + OS elicitation was carried out as described above. The procedure was carried out every other day for 6 d. Non-elicited plants and plants treated with lanolin paste without IAA or TCA were used as controls (n =10). The regrowth capacity after shoot removal of asGAL83, irAOC and EV plants after the mentioned treatments was monitored as described previously. IAA and TCA concentrations were chosen following previous studies (Baldwin et al., 1997; An et al., 1999).
Chlorophyll content of induced shoots
To determine whether the decrease in soluble sugar contents following leaf herbivory could be explained by a reduction in chlorophyll, we determined chlorophyll concentrations in induced leaves of EV and irAOC plants. Treatments were performed as described above (n =17). The chlorophyll content was quantified using a portable chlorophyll meter (SPAD 502; Konica Minolta, Tokyo, Japan).
Herbivore resistance of regrowing leaves
To evaluate whether regrowing leaves of previously infested plants were more resistant to subsequent attack, we measured M. sexta larval performance on regrowing leaves of previously treated plants. EV and irAOC plants were treated as described above (n =17), and one M. sexta neonate per plant was placed on the regrowing leaves 6 d after shoot removal and left to feed freely for 10 d. Larval mass was determined using a microbalance (Sartorius TE214S; Data Weighing Systems Inc., Elk Grove, IL, USA).
All statistical tests were carried out with Sigma Plot 12.0 (Systat Software Inc., San Jose, CA, USA) using analysis of variance (ANOVA). Levene's and Shapiro–Wilk tests were applied to determine error variance and normality. Fitness parameters to determine the effect of M. sexta attack on regrowth capacity were tested in a one-way ANOVA and Dunn's post-hoc tests. Fitness parameters of regrowing EV, irAOC and asGAL83 plants, as well as metabolite reconfiguration before and after shoot removal, were tested individually for each genotype in a one-way ANOVA and Holm–Sidak post-hoc tests. A non-parametric Kruskal–Wallis one-way ANOVA on ranks was carried out for variables that did not conform to normality. Two-way ANOVA and Holm–Sidak post-hoc tests, with treatment and genotype as factors, were used to determine the effect of simulated M. sexta attack on regrowth capacity in the field, fitness changes of regrowing grafted plants, chlorophyll contents after herbivore attack, larval performance on regrowing leaves, the contribution of stems to regrowth capacity and the role of GAs in regrowth. Two-way ANOVA and Holm–Sidak post-hoc tests were carried out to assess herbivory-induced phytohormone levels, with time and treatment as factors, for each time point individually, as well as to evaluate the role of auxin in regrowth capacity, with treatment and OS elicitation as factors. Fitness parameters to determine the effect of simulated M. sexta attack (W + OS) and soil moisture characteristics on regrowth capacity were assessed in a three-way ANOVA and Holm–Sidak post-hoc tests, with treatment, water condition and genotype as factors. Correlations between JA signaling and regrowth capacity were tested using Pearson product moment tests.
M. sexta attack constrains N. attenuata regrowth and fitness in the glasshouse and the field
Leaf-herbivore-induced C reallocation to the roots has been suggested as a potential tolerance mechanism in N. attenuata (Schwachtje et al., 2006). However, we observed that M. sexta attack reduces regrowth from the roots. Shoots regrowing from rootstocks of M. sexta-attacked plants had smaller rosettes, shorter branches and produced fewer flowers than regrowing shoots of control plants (Fig. 1a–e). Similar effects were observed for wounded plants treated with M. sexta oral secretions (W + OS) in the field (Fig. 1f–h). In contrast with EV plants, no differences were observed between the W + OS and control treatment in JA-deficient irAOC plants (Fig. 1g), suggesting that JA-dependent induced defenses might constrain regrowth.
Caterpillars typically only remove the leaves and rarely feed from stems. To confirm the patterns of the somewhat artificial shoot removal treatment (which aimed to specifically evaluate the contribution of rootstocks to regrowth) in a more realistic set-up, we defoliated plants without removing the stem. Plants with intact stems produced significantly more flowers at the end of the flowering period than plants that were regrowing from the rootstocks (Fig. S1). However, W + OS induction decreased flower production of regrowing plants independent of the type of shoot removal (Fig. S1), suggesting that our shoot removal treatment yields biologically meaningful data.
Roots supply resources for leaf regrowth
To understand whether resources can be mobilized from the roots to support shoot regrowth, we performed an experiment in complete darkness. This enabled us to determine shoot biomass accumulation in the absence of any photosynthetic activity. We found that N. attenuata plants were able to regrow from the rootstock in complete darkness. Both the remaining shoot and the regrowing leaves gained a significant amount of biomass (Fig. S2b,c). At the same time, root biomass was reduced, implying resource remobilization from belowground tissues (Fig. S2a). The aboveground plant parts accumulated c. 30 mg of dry matter, whereas the roots lost 100 mg. W + OS pretreatment reduced the biomass of the regrowing leaves by > 50%, whereas the reduction in root biomass was not altered significantly (Fig. S2a,c). Six days of W + OS treatment did not reduce root biomass compared with untreated controls (Fig. S2a), suggesting that W + OS treatment reduced root quality or conversion efficiency to support leaf regrowth.
Water stress improves plant regrowth capacity in a herbivore-independent manner
Water shortage is an important determinant of a plant's capacity to regrow as it changes C reallocation patterns (Geiger & Servaites, 1991) and co-occurs with herbivore attack of N. attenuata in nature (Schwachtje et al., 2006). We found that both EV and irAOC plants that were subjected to water stress for 6 d regrew significantly better from the rootstock. However, water stress did not affect the herbivory-induced reduction of regrowth (Fig. 2). Although W + OS treatment of EV plants reduced rosette diameter, branch length and number of flowers of regrowing plants, wounding of the leaves with a pattern wheel and application of water to the wounds (W + W) did not affect either branch length or flower production (Fig. 2), demonstrating that herbivore-associated molecular patterns are important to trigger the plant response. Again, irAOC plants did not display any significant changes in regrowth following either W + W or W + OS treatment.
Herbivore-induced constraints in regrowth are GAL83 and JA dependent
An earlier study demonstrated that the herbivore-induced down-regulation of GAL83 increases C allocation to the roots and improves flower production in herbivore-attacked N. attenuata plants in a JA-independent manner (Schwachtje et al., 2006). We therefore tested whether silencing this gene changes the regrowth patterns from the rootstock after simulated herbivory. Indeed, in contrast with EV plants, asGAL83 plants did not suffer from any fitness consequences on W + OS treatment (Fig. 3). To further understand the role of JA in the system, we also included irAOC plants and MeJA applications. Contrary to W + W and W + OS, MeJA treatment reduced regrowth in irAOC plants (Fig. 3).
Leaf- and root-derived JA constrain herbivore-induced regrowth
JA have been shown to regulate plant stress responses, growth and development (reviewed by Wasternack & Hause, 2013) in a tissue-specific manner (Nalam et al., 2012). By employing a grafting protocol to create chimeric plants that were either silenced in root (EV/irAOC) or root and leaf (irAOC/irAOC) JA production, we investigated the contributions of leaf- and root-derived JA to regrowth. Similar to our previous observations on non-grafted plants, W + OS-treated EV/EV plants had smaller rosettes, smaller branches and fewer flowers than control EV/EV plants (Fig. 4). No induction effect was observed in irAOC/irAOC plants. W + OS-treated EV/irAOC plants showed a reduction in shoot regrowth from the rootstock relative to non-treated EV/irAOC plants (Fig. 4). Interestingly, the relative difference between control and W + OS-treated plants was intermediate between EV/EV and irAOC/irAOC plants (Fig. S3), suggesting that both root and leaf JA contribute to the reduction in regrowth capacity.
Simulated herbivory reconfigures the primary and secondary metabolism of shoots and roots in a Gal83- and JA-dependent manner
The induction of defense on herbivory attack might deplete plant resources that could otherwise be used to regrow. To test this hypothesis, we profiled soluble sugars and starch in herbivore-attacked plants. Six days after W + OS induction, concentrations of starch and soluble sugars were strongly reduced in EV leaves and roots (Fig. 5a), suggesting resource depletion. Nicotine, however, was induced in roots after OS elicitation (W + OS) and in leaves after both W + W and W + OS treatments (Fig. 5b). asGAL83 plants showed reduced levels of starch and elevated levels of nicotine in wounding and water (W + W)-treated leaves, but no effect of the application of oral secretions was observed (Fig. 5a,c). Accordingly, sugar contents in the leaves and roots of asGAL83 plants remained unaltered (Fig. 5b). irAOC plants did not display any changes in sugars and nicotine, and starch levels were only reduced in wounded leaves (Fig. 5a–c). MeJA application to irAOC plants restored EV patterns (Fig. 5a–c). Taken together, these results show that the induction of nicotine and the depletion of carbohydrates in the roots are positively correlated, and that root C depletion is correlated with a reduction in regrowth from the rootstock. The regrowing leaves of EV and asGAL83 plants did not differ in starch and sugar concentrations (Fig. 5d,e). Nicotine levels were increased more strongly in EV than in asGAL83 plants (Fig. 5f). Regrowing leaves in irAOC plants had higher levels of starch on W + W and W + OS pretreatment, whereas nicotine levels were reduced (Fig. 5d–f). This pattern was restored to wild-type levels on MeJA application (Fig. 5d–f). This suggests that regrowing leaves are better defended, but potentially also more costly to produce, for EV plants.
Foliar herbivory induces JA and IAA in the roots and leaves
Systemic signals may be required to trigger the herbivory-induced reduction in regrowth from the rootstock. We therefore evaluated the induction of phytohormones in response to M. sexta attack. As demonstrated previously, JA and (+)-7-iso-jasmonoyl-l-isoleucine (JA-Ile) were up-regulated within 1 h after wounding of EV plants (Fig. 6a–f). The application of oral secretions (W + OS) amplified this response. The JA burst was also observed in asGAL83 plants, even though induced JA-Ile levels were slightly lower. JA were not induced in irAOC leaves. Neither JA nor JA-Ile were up-regulated in the roots of EV and irAOC plants. However, JA-Ile was up-regulated in the roots of asGAL83, 3 h after W + OS treatment. Within 1 h after leaf elicitation, IAA was highly up-regulated in the leaves and roots of all genotypes (Fig. 6g–i). IAA levels were maintained above controls in W + OS-treated asGAL83 and irAOC plants over 3 h, whereas they dropped to control levels in EV plants 1 h after elicitation. ABA levels remained unaffected in the roots and were slightly induced in the leaves (Fig. S4a–c). Salicylic acid (SA) was highly induced in the leaves 3 h after treatment in all genotypes. SA levels in the roots remained unaffected (Fig. S4d–f), apart from a slight increase in asGAL83 plants 1 h after W + OS treatment. Taken together, these results show that the JA burst is conserved in asGAL83 plants and that auxin may act as a systemic leaf-to-root signal in N. attenuata.
JA signaling and regrowth capacity are negatively correlated in natural accessions
To confirm the role of JA in determining the regrowth responses among different natural N. attenuata populations, we measured both parameters in 120 field-collected individuals from different populations. We found a significant negative correlation between W + OS-induced JA production and flower production of regrowing shoots (Pearson's correlation r =−0.217, P =0.0182; Fig. 7). The relatively low correlation coefficient suggests the presence of JA-independent regulatory elements that determine regrowth patterns.
Little evidence for the involvement of GAs in regrowth responses
Plants seem to prioritize defense over growth via an antagonistic crosstalk between JA and GA signaling (Yang et al., 2012). Monitoring of the changes in branch architecture as a GA signaling downstream marker, however, provided little evidence for the involvement of GAs in root responses to insect attack. W + OS-treated EV plants had shorter branches and fewer internodes than, but similar internode lengths to, control plants 18 and 20 d after shoot removal (Fig. S5a,d,g,j). W + OS elicitation did not affect significantly the branch architecture of asGAL83 plants, apart from a slight reduction in the average internode length 20 d after shoot removal (Fig. S5b,e,h,k). Simulated herbivory did not affect the branch architecture of irAOC plants (Fig. S5c,f,i,l).
IAA application restores wild-type patterns in asGAL83, but not in irAOC, plants
Nicotiana attenuata responds to M. sexta herbivory by inducing auxin levels in both leaves and roots. To explore the possible role of auxin homeostasis in tolerance responses, we applied IAA or TCA to alter auxin transport and monitored plant regrowth following elicitation. The application of the auxin transport inhibitor trans-cinnamic acid to EV plants attenuated the fitness cost of simulated M. sexta herbivory (Fig. 8a,d,g). IAA application, however, did not change the regrowth pattern of EV plants. However, IAA application to asGAL83 plants resulted in an increase in regrowth from the rootstock in controls, but not in W + OS-treated plants. As a consequence, IAA-supplemented asGAL83 plants behaved like EV plants in terms of their herbivore-induced regrowth patterns (Fig. 8b,e,h). TCA application also increased the growth of asGAL83 plants. irAOC plants were not influenced by either IAA or TCA application (Fig. 8c,e,i). Taken together, these experiments suggest a role for IAA in determining the herbivory-induced regrowth patterns in EV and irGAL83 plants.
Attacked leaves have lower chlorophyll contents
The reduction in non-structural carbohydrates in herbivore-attacked plants might be explained by an increase in energy demand or a decrease in photosynthetic energy supply. We therefore measured chlorophyll content as an indication of photosynthetic capability. Overall, chlorophyll contents did not differ between EV and irAOC plants (Fig. S6). Wounding reduced the chlorophyll contents of the treated leaves in both genotypes. The application of oral secretions further reduced chlorophyll levels in EV, but not in irAOC, plants. These results indicate that, in addition to the energy required for the biosynthesis of defensive compounds, reduced photoassimilation may contribute to the herbivory-induced JA-dependent depletion of carbohydrates.
JA signaling determines the induced resistance of regrowing leaves
Our previous results suggested that regrowing shoots of previously attacked plants might be better defended (Fig. 5f) and therefore more costly to produce. To further evaluate the defensive status of regrowing leaves, we measured M. sexta performance on regrowing pretreated plants. Overall, caterpillars performed better on regrowing shoots of irAOC than EV plants. Plants growing from EV rootstocks that had previously been subjected to simulated herbivory supported less M. sexta growth than untreated plants (Fig. S7). Surprisingly, M. sexta performed better on regrowing shoots of irAOC plants that had been wounded before. No effect of W + OS treatment was observed in irAOC plants, whereas MeJA treatment reduced M. sexta performance (Fig. S7).
In this study, we provide evidence for a role of roots in resource-based trade-offs between resistance and tolerance to herbivory. We found that simulated M. sexta herbivory reduces starch and sugar contents and induces nicotine production in the roots and leaves of N. attenuata. The reduction of non-structural carbohydrates in roots was correlated with a reduction in regrowth from the rootstock in both the glasshouse and the field. In many plants, an increase in C transport from both damaged and undamaged tissues to the roots has been observed (Dyer et al., 1991; Briske et al., 1996; Holland et al., 1996; Babst et al., 2005, 2008; Bazot et al., 2005; Schwachtje et al., 2006; Kaplan et al., 2008; Gómez et al., 2010, 2012; Ferrieri et al., 2012). Although it has been proposed that the herbivory-induced resource sequestration acts as a putative tolerance mechanism by increasing root reserves for future regrowth (Schwachtje et al., 2006), the lack of evidence for an actual accumulation of carbohydrate resources in the roots has called this view into question (Steinbrenner et al., 2011; Gómez et al., 2012). In our experiments, we expected that induced resource sequestration would increase the capacity of N. attenuata to regrow after shoot removal. However, in none of the investigated plant genotypes and environmental conditions was such an effect observed. Although Schwachtje et al. (2006) demonstrated that silencing GAL83 increases C allocation to the roots and prolongs flowering in N. attenuata, that study did not provide any direct evidence for herbivore-induced plant tolerance via increased C storage. Early flower production in wild-type plants was reduced on elicitation (Schwachtje et al., 2006). Our results confirm this finding and show that the down-regulation of GAL83 improves regrowth on herbivory. It is noteworthy that C reallocation documented by Schwachtje et al. (2006) was independent of JA, whereas the C depletion and regrowth patterns in this study were influenced by JA. Based on this evidence, we propose that M. sexta-induced resource sequestration in N. attenuata does not increase root C storage and defoliation tolerance per se, but may support the synthesis of plant defensive metabolites below ground (Shoji et al., 2000) and/or buffer against a breakdown of root primary functions in the face of carbohydrate depletion. A good mechanistic understanding of resource allocation processes and C fluxes will be necessary to test these hypotheses in a more comprehensive manner in the future. In this context, it will also be important to compare regrowth patterns across different plant species. Nicotiana attenuata synthesizes large amounts of nicotine in the roots which are then transported to the leaves. Plants that do not directly engage their roots in defensive processes may show different regrowth patterns.
Our study demonstrates that JA play a central, but not exclusive, role in root-mediated growth–defense trade-offs: Contrary to wild-type plants, JA-deficient irAOC plants did not suffer from herbivory-induced reduction of root carbohydrates and were not impaired in their regrowth capacity in the glasshouse and the field. Furthermore, the induction of defenses was abolished in irAOC plants, and the regrowing leaves were more susceptible to M. sexta attack. MeJA application restored wild-type patterns in irAOC plants. The central role of JA as determinants of the defensive make-up in nature is illustrated by the fact that the JA burst is negatively correlated with defoliation tolerance across field-collected N. attenuata genotypes. A reduction in the herbivore-induced suppression of leaf growth has also been documented in JA-deficient asLOX3 plants (Zavala & Baldwin, 2006), and it has been proposed that a JA-dependent reduction in leaf photosynthesis may be responsible for this effect (Nabity et al., 2009, 2013). In accordance with this hypothesis, we found that simulated herbivory reduces leaf chlorophyll concentrations in a JA-dependent manner. It is therefore likely that JA signaling depletes the plant's C pools by inducing the production of defensive metabolites on the one hand and reducing photoassimilation on the other. Even though our micrografting approach suggested that the aboveground JA burst is sufficient to trigger a reduction in the plant's regrowth capacity from the rootstock, we found that plants silenced in root JA production displayed an intermediate regrowth phenotype. It is therefore likely that the de novo synthesis of JA in the roots (Wang et al., 2008; Bonaventure et al., 2011) also contributes to the regulation of plant tolerance, for example by contributing to induced nicotine biosynthesis.
Resource-based trade-offs between growth and defense have long been discussed (McKey, 1974; van der Meijden et al., 1988; de Jong & van der Meijden, 2000; Schwachtje & Baldwin, 2008; Anten & Pierik, 2010; Orians et al., 2011) and hormonal cross-talk has been proposed as a possible mechanism (Chen et al., 2011; Yang et al., 2012). A recent study in rice and A. thaliana proposed that JA may reduce plant growth by interfering with the GA-mediated promotion of internode elongation (Yang et al., 2012). We found little morphological evidence of JA/GA crosstalk in regrowing shoots, and propose that the depletion of storage carbohydrates, rather than a hormone-dependent reduction in cellular activity, may restrain N. attenuata regrowth. Nevertheless, the relatively weak correlation between JA production and regrowth among natural accessions suggests that regulatory elements other than JA may influence the plant's root storage regime and regrowth capacity. One prominent candidate in this context is IAA, which has been proposed as a negative regulator of nicotine biosynthesis and JA accumulation in Nicotiana sp. (Baldwin et al., 1997; Shi et al., 2006; Onkokesung et al., 2010). In contrast with our expectations, we found that IAA rapidly accumulated in the leaves and roots of herbivore-attacked N. attenuata plants, and that the root auxin response was prolonged in both asGAL83 and irAOC lines. The fact that the inhibition of auxin transport reduced the herbivore-induced reduction in regrowth in EV plants, whereas IAA application restored wild-type patterns in asGAL83 plants, strongly suggests that auxin homeostasis is an important determinant of plant tolerance against herbivory.
Our understanding of how plants coordinate and fine tune JA and IAA signaling is still limited. Glucose signaling has been proposed to coordinate plant growth by interfering with auxins (Moore et al., 2003; Sairanen et al., 2012), and SNF-related serine/threonine-protein kinases (SnRK kinases) can regulate carbohydrate partitioning (reviewed by Halford & Paul, 2003; Rolland et al., 2006) and mediate the binding of the 26S proteasome to SCF ubiquitin ligases (Farras et al., 2001), suggesting that the SnRK1 kinases promote auxin signaling via the activation of auxin-responsive gene transcription. Furthermore, the A. thaliana auxin-responsive genes GH3.3, GH3.5 and GH3.6 have been found to conjugate jasmonic acid to amino acids, leading either to JA degradation or affecting the synthesis of JA-Ile (Gutierrez et al., 2012). Therefore, it is conceivable that a tight interplay between root/shoot auxin ratios and GAL83-mediated resource partitioning coordinates growth and root defenses in N. attenuata in addition to JA. Further experiments involving a tight spatio-temporal control of auxin and carbohydrate fluxes will be necessary to disentangle the exact mechanisms in detail.
In conclusion, we have shown that herbivore attack results in the depletion of non-structural carbohydrates in the roots which is likely to constrain the plant's capacity to regrow and, at the same time, enable the deployment of effective defenses. Both JA and IAA regulate trade-offs between induced defenses and tolerance. The underlying regulatory network is likely to provide a robust mechanistic basis for the divergent intraspecific strategies that plants display to survive in a hostile environment.
We thank the Max Planck Society for funding and Brigham Young University for the use of the Lytle Ranch Preserve, C. Diezel, D. Kessler and P. Kumar for help with field experiments, M. Reichelt with the phytohormone measurements and P. Bonilla, J. Lu and R. Ramakrishnan with the glasshouse experiments. M.E. and C.A.M.R. are supported by a Marie Curie Intra European Fellowship (grant no. 273107 to M.E.) and a Swiss National Foundation Fellowship (grant no. 140196 to C.A.M.R.).