Induced carbon reallocation and compensatory growth as root herbivore tolerance mechanisms

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1 bs_bs_banner Plant, Cell and Environment () 7, 6 6 doi:./pce.59 Original Article Induced carbon reallocation and compensatory growth as root herbivore tolerance mechanisms Christelle A. M. Robert,, Richard A. Ferrieri, Stefanie Schirmer,, Benjamin A. Babst, Michael J. Schueller, Ricardo A. R. Machado,, Carla C. M. Arce,,5, Bruce E. Hibbard 6, Jonathan Gershenzon, Ted C. J. Turlings 7 & Matthias Erb,8 Root-Herbivore Interactions Group, Departments of Biochemistry and Molecular Ecology, Max Planck Institute for Chemical Ecology, Hans-Knöll-Str. 8, 775 Jena, Germany, Bioscience Department, Brookhaven National Laboratory, Upton, NY 97, USA, 5 Departamento de Entomologia, Universidade Federal de Viçosa, Viçosa, Minas Gerais, Brazil, 6 Plant Genetics Research Unit, USDA-ARS, University of Missouri, 5 Curtis Hall, Columbia, MO 65, USA, 7 Laboratory for Fundamental and Applied Research in Chemical Ecology (FARCE), University of Neuchâtel, Rue Emile Argand,, CH- Neuchâtel, Switzerland and 8 Institute of Plant Sciences, University of Bern, Altenbergrain, CH- Bern, Switzerland ABSTRACT Upon attack by leaf herbivores, many plants reallocate photoassimilates below ground. However, little is known about how plants respond when the roots themselves come under attack. We investigated induced resource allocation in maize plants that are infested by the larvae Western corn rootworm Diabrotica virgifera virgifera. Using radioactive CO, we demonstrate that root-attacked maize plants allocate more new C carbon from source leaves to stems, but not to roots. Reduced meristematic activity and reduced invertase activity in attacked maize root systems are identified as possible drivers of this shoot reallocation response. The increased allocation of photoassimilates to stems is shown to be associated with a marked thickening of these tissues and increased growth of stem-borne crown roots. A strong quantitative correlation between stem thickness and root regrowth across different watering levels suggests that retaining photoassimilates in the shoots may help rootattacked plants to compensate for the loss of belowground tissues. Taken together, our results indicate that induced tolerance may be an important strategy of plants to withstand belowground attack. Furthermore, root herbivoreinduced carbon reallocation needs to be taken into account when studying plant-mediated interactions between herbivores. Key-words: Diabrotica virgifera; CO ; compensatory root growth; plant herbivore interactions. INTRODUCTION Plants are able to perceive and respond to a broad spectrum of biotic and abiotic stimuli in an integrated manner, which allows them to optimize resource allocation, and ultimately, fitness, in a continuously changing environment (Karban Correspondence: M. Erb, Institute of Plant Sciences, University of Bern, Altenbergrain, Bern, Switzerland. et al. 997; van Dam 9b). Defensive strategies against herbivores include resistance traits that repel, deter or kill the attacker (Howe & Jander 8) as well as tolerance mechanisms that allow regrowth and reproduction after tissue loss (Strauss & Agrawal 999). Although resistance mechanisms have been extensively studied, much less is known about the mechanistic basis of tolerance (Stowe et al. ). Tolerance to herbivory typically relies on the activation of dormant meristems, the increase of photosynthetic activity and the diversion of resources away from the attacked tissues into storage organs that are inaccessible to foraging herbivores (Strauss & Agrawal 999; Schultz et al. ). Resource reallocation following real or simulated leaf attack has been documented in numerous plant species, including tomato, tobacco, maize, barley and poplar, all of which increase the export of photoassimilates from the leaves to the stem and roots upon herbivory (Holland et al. 996; Hochwender et al. ; Babst et al. 5, 8; Schwachtje et al. 6; Henkes et al. 8; Gómez et al. ; Hanik et al. a). Similarly, nitrogen allocation to the roots was found to increase in tomato plants that were subjected to simulate aboveground herbivory (Gómez et al. ). Yet, shifts in resource allocation do not necessarily coincide with induced tolerance per se. For instance, free amino acids allocated to the leaves of tobacco plants are used for the biosynthesis of defensive secondary metabolites rather than regrowth (Hanik et al. b). Furthermore, increased resource flow to the roots can lead to more carbon exudation into the rhizosphere, with no net storage of assimilates (Holland et al. 996). One of the few examples where herbivore-induced resource reallocation was correlated with tolerance comes from wild tobacco (Nicotiana attenuata), where silencing of herbivoresuppressed SNF-like kinase delayed senescence and prolonged flowering under water deprivation (Schwachtje et al. 6). However, a recent study found that regrowth from rootstock of N. attenuata was reduced by prolonged herbivore attack, an effect that coincided with root carbon depletion (Machado et al. ). A herbivore-induced reduction of root carbohydrates has also been found in tomato (Gómez John Wiley & Sons Ltd 6

2 6 C. A. M. Robert et al. et al. ). However, in contrast to wild tobacco, induced tomato plants were found to regrow better after defoliation than control plants (Korpita et al. ). From these studies, it becomes evident that understanding plant tolerance to herbivory requires a thorough evaluation of the mechanisms behind resource partitioning and allocation to storage and defence (Orians et al. ). A largely neglected aspect of tolerance-related resource diversion below ground is the fact that roots may not be a safe haven for photoassimilates after all because they are under constant attack by various consumers including insects, nematodes and micro-organisms (van Dam 9a; Orians et al. ). How do plants reallocate resources when their roots are under attack? Using a 5 N labelling approach, it was demonstrated that the spotted knapweed Centaurea maculosa increases relative N allocation to the shoot upon attack by the root-boring larvae of Agapeta zoegana (Newingham et al. 7). This reallocation response was interpreted as a possible compensatory response that would allow the plant to maintain essential processes in the shoot under root herbivore-induced N limitation (Newingham et al. 7). So far, the only study that investigated carbon partitioning in root herbivore-attacked plants found no clear patterns of C allocation in Diabrotica virgifera attacked maize plants at the reproductive stage, apart from a slightly higher allocation to the husks (Xue et al. ). D. virgifera larvae commonly attack plants at the vegetative stage (Hibbard et al. 8), and it remains unknown how maize responds to root herbivory in this context. A potentially important parameter that may influence root herbivore-induced carbon reallocation is the plant s water status. Root herbivores can induce water stress in the shoots (Masters et al. 99), which can trigger abscisic acid (ABA) signalling (Erb et al. 9) and stomatal closure, ultimately resulting in a reduction in photosynthetic activity (Dunn & Frommelt 998). In many cases, root herbivore-induced water stress is more likely if water supply in the soil is limiting (Dunn & Frommelt 998; Erb et al. ), and it can therefore be expected that soil humidity may modulate carbon allocation patterns. Based on the fact that D. virgifera tolerant maize genotypes that are able to compensate for belowground tissue loss by growing additional roots (Prischmann et al. 7), we hypothesized that maize should possess effective inducible tolerance mechanisms, including, for instance, carbon reallocation away from the attacked roots to support regrowth. Using radioactive CO, we explored the dynamics of carbon partitioning between leaves and roots following root herbivory. In addition, we performed a number of chemical and biochemical analyses and growth assays to link the observed carbon reallocation pattern to compensatory root growth responses. Although the currently available bioassays make it difficult to distinguish between herbivore-specific and general root damage responses (Erb et al. ), we aimed at disentangling the two by correlating root damage and root regrowth. Taken together, our experiments shed light on carbon reallocation as a potential plant tolerance strategy against root damage by herbivores. MATERIALS AND METHODS Plant and insect growth Maize plants (Zea mays L., variety Delprim) for the radiolabelling experiments were germinated on wet filter paper in Petri dishes for d. The seedlings were then transplanted into cylindrical glass cells (7 mm ID 5 mm length; QGlass Co, Towaco, NJ, USA) containing an agarose growth medium that was obtained by adding.6 g of Hoagland modified basal salt mixture (PhytoTechnology Laboratories TM, Shawnee Mission, KS, USA) and.55 g of -(N-morpholino) ethanesulfonic acid (MES) hydrate (Sigma Life Science) to L of distilled water. After adjusting the ph to 5.8 with a few droplets of sodium hydroxide,.5 g of Gelzan TM CM (Sigma Life Science, St. Louis, MO, USA) was added. All media and glass cells were autoclaved prior to use. Plants were grown under metal-halide lamps ( ± C, 6% relative humidity, 6:8 h L/D, and 5 μmol m s ) and used for experiments when they were approximately weeks old and had three to four fully developed leaves. Maize plants for regrowth and tolerance experiments were sown in L of plastic pots (Pöppelmann, Lohne, Germany) by placing the seeds on a layer of moist washed sand ( mm grain size; Raiffeisen, Germany) that was then covered with cm of commercial soil (Tonsubstrat, Geeste, Germany; Aussaaterde, Ricoter, Aarberg, Switzerland). Seedlings were grown in a greenhouse ( ± C, 6% relative humidity, 6:8 h L/D). Ferty fertilizer (Ferty, Planta GmbH, Regenstauf, Germany) was added twice a week after seed germination. Eggs of D. virgifera virgifera LeConte (Coleoptera: Chrysomelidae) were obtained from the USDA-ARS Columbia (MO, USA) and USDA-ARS- NCARL Brookings (SD, USA). After hatching they were kept on freshly germinated maize roots until the start of the experiments. Second instar larvae were used for all induction treatments. Radiotracer production and administration To determine changes in carbon allocation patterns in D. virgifera infested plants, we performed a set of pulse-chase experiments using radioactive CO. This tracer was produced via the N(p, α) C nuclear transformation (Ferrieri & Wolf 98) from a ml target filled with high-purity nitrogen gas ( ml at standard temperature and pressure) using 8 MeV protons from the TR-9 (Ebco Industries Ltd, Richmond, BC, Canada) cyclotron at Brookhaven National Laboratory, and captured on a molecular sieve ( Å). The CO that was trapped on the molecular sieve was desorbed and quickly released as a pulse of tracer into an air stream possessing an ambient concentration of CO and flowing at a rate of ml min. This stream of air was directed into a lighted 5 cm cell (9 μmol m s ) maintained at C in which a portion of a source leaf was affixed (Ferrieri et al. 5). The load leaf affixed within this cell was continuously fed a supply of ambient air both during the passage of the CO pulse and afterwards for h while whole-plant dynamics were acquired. A PIN diode radiation detector (Carroll John Wiley & Sons Ltd, Plant, Cell and Environment, 7, 6 6

3 Induced root herbivore tolerance 65 Ramsey Associates, Inc, Berkeley, CA, USA) affixed to the bottom of the leaf cell enabled continuous measurement of radioactivity levels within the cell during and after the pulse. These measurements were facilitated by online computer acquisition using Peak Simple.56 software (SRI Instruments, Torrance, CA, USA) interfaced with an SRI acquisition system (SRI Instruments, Torrance, CA, USA). Metrics from this one detector included quantification of leaf fixation of CO and quantification of leaf export of C- photoassimilates. Because the CO tracer arrives in the leaf cell as a discrete pulse (fwhm < min), we were able to use the difference between the pulse height radioactivity level and the leaf radiation level to calculate a fractional fixation value. We note that even though gas exchange data were not collected during these tracer studies, we have previously shown that CO fixation values positively correlate with photosynthetic rates (Ferrieri et al. 5). Furthermore, as a C plant, maize avoids photorespiration under normal growth conditions, enabling us to exclude this parameter from the analysis. Transport and allocation of C-photoassimilates To profile carbon allocation, maize plants were either infested with D. virgifera larvae for d, or left uninfested, prior to radiotracer exposure. - mci of CO were administered to an intact source leaf of each study plant, and the PIN detector was used to record CO administration, tissue fixation and leaf export of C-photoassimilates. Two hours after tracer administration, shoots were excised and the whole root system was carefully removed from the agar growth medium. Fresh weights of these plant tissues were immediately measured. The amount of C radioactivity present in roots, shoots (comprising both labelled and nonlabelled leaves and stems), growth medium and D. virgifera larvae was quantified using a combination of gamma counting instrumentation including a Capintec Radioisotope Dose Calibrator CRC- (Capintec, Inc, Ramsey, NJ, USA) for measuring micro- to millicurie levels and a NaI scintillation detector for measuring sub-microcurie levels of radioactivity. Radioactivity was corrected for decay using the end-ofbombardment time as time zero. Detailed visualization C- photoassimilate distribution within individual leaves, roots, meristems and larvae was obtained by exposing phosphor plates to the radioactive tissues and radioactive larvae and using positron autoradiography (Fuji BAS-5 Imager Fujifilm Imaging, Stamford, CT, USA) coupled with Science Laboratory 99 Image Gauge software (Fuji Photo Film Co, Ltd, Tokyo, Japan). Using this approach, infested and control plants were measured in a pairwise design (n =, one control and one infested plant per day). Stem morphology Stems are potential sinks for leaf assimilates (Scofield et al. 9). To investigate whether D. virgifera infestation changes this tissue, we performed an experiment to investigate John Wiley & Sons Ltd, Plant, Cell and Environment, 7, 6 6 whether root infestation modifies stem morphology of maize seedlings. Three-week-old maize seedlings were infested with six second instar larvae for 7 d (n = 8). Larval density and infestation time of the soil-grown plants was chosen to match damage levels of the CO labelling experiment (see Results section). At the end of the experiment, stem height, circumference and mass were recorded. Furthermore, the stem density (g cm ) was determined using the cylindrical dimensions to approximate stem volume. Invertase activity and carbohydrate profiles To investigate how D. virgifera attack influences carbohydrate pools and the activity of sucrose-cleaving enzymes, -week-old seedlings were infested with six D. virgifera larvae as described above. plants remained uninfested (n = ). Stem circumference was determined every d. Seven days after infestation, the root systems of the plants were excavated, cleaned under a running stream of water for about s and then flash-frozen in liquid nitrogen. The stems and leaves of half the plants were harvested and flash-frozen (n = 6), whereas the other half was kept for regrowth monitoring (see below). All tissues were weighed before freezing. Starch levels in the roots, stems and leaves of control and D. virgifera infested plants were determined enzymatically as described in Machado et al. (). Sucrose, glucose and fructose concentrations were measured with high-performance liquid chromatography triple quadruple mass-spectrometry (HPLC/MS-MS) as described (Falk et al. ). The activity level of the soluble and insoluble invertase components was determined according to previously established protocols (Ferrieri et al. ). To determine the dry weight and water contents of the different tissues, mg of pulverized material per sample was lyophilized for 7 h and re-weighed. Root regrowth To investigate the regrowth capacity of D. virgifera attacked plants, shoots of control and root-infested plants from the experiment above (n = 6) were placed in individual water beakers (5 ml of Erlenmeyer flasks filled with ml of tap water, covered with aluminium foil). The water in the beakers was replenished every 6 7 d. The number of emerging crown roots was then counted over a period of weeks. At the end of the experiment, stem circumference was measured. Effect of water supply on root regrowth To investigate the regrowth capacity of D. virgifera attacked plants under different watering regimes, -week-old seedlings were infested as described above (n = ). Each plant was potted individually and placed in an individual plastic dish (Platecnic ; Perego, Italy). Plants were watered daily with 5, or ml of water. In order not to wash the larvae out of the root system during watering, all plants were

4 66 C. A. M. Robert et al. first watered with 5 ml from the top, and the complementary 5 and 5 ml of the ml and ml watering treatment were added to the dish below the pots. These treatments resulted in different moisture levels in the pots without any visual water stress symptoms for the plants. After 7 d of infestation, stem circumference was measured, the plant root systems were gently washed in water and all of the D. virgifera larvae were cautiously removed. Root damage was evaluated using Oleson s scale (Oleson et al. 5), and the last generation of crown roots was cut from all plants to standardize the regrowth observations for control and infested plants. The plants were then transplanted into individual pots ( L; Pöppelmann, Lohne, Germany) filled with soil (Tonsubstrat, Geeste, Germany) and watered daily by immersion in cm tap water for 5 min. Regrowing crown roots were counted 7 and d after repotting by gently removing the soil around the stem until the base of the regrowing roots became visible. After d, the newly grown roots were harvested, dried for 8 h at 8 C, and weighed. Statistical analyses All statistical analyses were performed using analysis of variance (anova) in the software package Sigmaplot. (Scientific Solution SA, Pully, Switzerland). Data were first analysed with Levene s and Kolmogorov Smirnov test to determine heteroscedasticity of error variance and normality. To account for the pairwise design in the CO labelling experiment, healthy and infested plants were compared using paired t-tests. In cases where the Levene s and Kolmogorov Smirnov tests indicated a violation of anova conditions, Wilcoxon signed-rank tests were used. Sugar and starch concentrations as well as invertase activities from fully randomized experiments were compared using two-way anovas followed by Holm-Sidak post hoc tests. Regrowth patterns were evaluated with repeated-measures anovas and two-way anovas. Data sets from fully randomized experiments that did not fulfil the assumptions for anovas were log or rank-transformed prior to analysis. µci fixed ( CO/cm ) Total allocationof C (mci) C accumulation in meristems (% root activity) (c) (e) Shoots Leaves Roots Exudates P =.6 Root infested Root infested Larvae C (shoot:root) Leaf export (%) 5 (d) Root infested Root infested Root infested Figure. Root herbivore attack increases shoot carbon allocation. Results from an CO labelling experiment involving plants without ( control ) and plants with Diabrotica virgifera infested root systems ( root infested ) are shown. CO amount fixed by the leaves. Shoot : root ratios of fixed C h after the labelling pulse. (c) Decay-corrected accumulation of Cin different plant organs, the rooting medium and the feeding larvae h after pulse labelling. (d) Amount of C exported from the pulsed leaves over h. (e) Relative accumulation of C label in the root meristems compared with the entire root system. Asterisks indicate significant differences between treatments (paired t-test, P <.5). Error bars represent standard errors (±SE). RESULTS Root herbivory increases carbon reallocation to the shoots To understand how root attack by D. virgifera changes carbon allocation patterns in vivo, we traced the flow of radioactive carbon administered as an CO pulse to the leaves of control and D. virgifera infested plants. Root attack by D. virgifera reduced shoot and root fresh mass by approximately % (Supporting Information Fig. S). Overall, CO fixation marginally increased in infested plants (paired t-test, t =.69, P =.6; Fig. a). In out of infested control plant pairs, D. virgifera infestation increased the amount of CO that was fixed within the load leaf (χ =.8; P =.). Gamma counting of tissues revealed that C- photoassimilate allocation was also affected by root herbivory. The shoot : root ratio increased by % in infested plants (paired t-test, t =.89, P =.; Fig. b), demonstrating that D. virgifera attack induces carbon reallocation to the shoot. More detailed measurements of radioactivity levels in the different compartments (including plant tissues, growth medium and larvae) confirmed this pattern. The absolute amount of C-photoassimilates was higher in the shoot of infested than control plants, whereas it was similar in roots and exudates (shoots: Wilcoxon signed-rank test, Z statistic =., P =.; roots: paired t-test, t =.6, P =.556; exudates: paired t-test, t =.6, P =.87; Fig. c). Interestingly, h after the administration of the CO pulse, we were already able to detect radioactive carbon in the matrix surrounding the roots and the feeding larvae, demonstrating that carbon is fixed, translocated to heterotrophic tissues (i.e. the roots) and transferred across trophic levels (i.e. into the surrounding matrix and the D. virgifera larvae) within a few hours. The larvae of infested plants were themselves a significant carbon sink (Fig. c), John Wiley & Sons Ltd, Plant, Cell and Environment, 7, 6 6

5 Induced root herbivore tolerance 67 even though they contained only a small fraction of the total C activity administered to the plant (Fig. c). Overall, infested plants exported more C-photoassimilates from the feeding leaves (n = ; Wilcoxon signed-rank test, Z statistic =.8, P =.; Fig. d).these results show that the increase in shoot carbon allocation is not due to higher accumulation in the feeding leaves themselves, but increased incorporation into tissues such as stems and other leaves. Root infested Root herbivory decreases root meristematic activity To gain further insight into how D. virgifera infestation changes root meristematic activity and photoassimilate accumulation, we imaged the full root systems and calculated the fractions of radioactivity that accumulated in the root tips. D. virgifera infestation significantly reduced the relative accumulation of radiotracers in the root tips (paired t-test, t =.9, P =.; Figs e & ), indicating a decrease in the sink strength of the root meristematic regions. Root herbivory decreases root invertase activity and increases carbohydrate pools Alteration of whole-plant carbon allocation patterns in response to herbivory can be the result of changes in invertase activity that alter sink strength in those tissues (Schwachtje et al. 6; Kaplan et al. 8). These responses can lead to longer term changes in sugar and starch pool sizes (Machado et al. ) (Gómez et al. ). To investigate whether D. virgifera attack influences these parameters in maize, we profiled the content of soluble sugars and starch, as well as sucrose conversion rates of soluble and insoluble invertases in the roots, leaves and stems of attacked plants. Note that the chosen infestation time and density resulted in a % decrease of root fresh mass (Supporting Information Fig. S) and was therefore comparable with the CO experiment. D. virgifera attack led to a decrease of both soluble and insoluble invertase activity in the roots, resulting in a significantly lower total invertase activity below ground (Holm- Sidak post hoc test: P =.; Fig. a). Starch levels were significantly different between tissues, with the highest amounts in the leaves, followed by stems and roots (two-way anova: P <.; Fig. b). Soluble sugars (glucose, fructose and sucrose) followed a similar distribution pattern (Fig. c). D. virgifera attack did not change starch pools (Fig. b), but significantly increased total soluble sugar levels across all tissues (two-way anova: P =.). On an individual sugar basis, this increase was only significant for sucrose in the leaves and roots (Holm-Sidak post hoc tests: P <.5; Fig. c). Root herbivory increases stem mass As root herbivory increased leaf fixation and leaf export of photoassimilates without increasing root allocation, we hypothesized that the stem may serve as a storage organ John Wiley & Sons Ltd, Plant, Cell and Environment, 7, 6 6 Figure. Root herbivore attack reduces belowground meristematic activity. Representative colour-inverted phosphorplate images of control (left) and Diabrotica virgifera infested roots (right) of CO -labelled plants are shown. High positron emissions from photoassimilate allocation-associated C decay result in dark staining. Roots of different individual plants. Image of an individual root with the primary root tip (), secondary root tips () and D. virgifera larvae that have fed on the same root (). The labelled carbon starts accumulating in the root herbivores only h after administering the CO pulse to leaves. Note that the exposure in figure was increased compared with composite figure to improve viewing quality. that accumulates excess carbon for compensatory growth of stem-borne roots. As a first marker for D. virgifera induced changes in stem physiology, we measured the stem circumference, height, weight and density of attacked plants. Seven days after infestation, D. virgifera attacked plants had shorter, but thicker stems (t-tests, P <.5; Fig. a,b). Stem mass lightly increased in root-infested plants (Fig. c). Stem density remained constant (Fig. d), indicating that the mass gain is not caused by a shift in dry mass accumulation.

6 68 C. A. M. Robert et al. Invertase activity (mg sucrose g FW min ) Starch (mg g FW ) Tissue:. Treatment:.6 TT:.95 Insoluble Soluble Tissue:<. Treatment:.6 TT:.7 Stem height (cm) Stem mass (g FW) (c) 6 Infested Infested Stem circumference (cm) (d) Infested Infested Figure. Root attack increases stem thickness and biomass. Stem height from the base to the first leaf node of control and root-infested plants. Stem circumference of control and root-infested plants. (c) Stem mass of control and root-infested plants. (d) Stem density of control and root-infested plants. Asterisks indicate significant differences (t-test: P <.5, P <., P <.). Error bars represent standard errors (±SE). Stem density (g FW cm ) Soluble sugars (mg g FW ) (c) Suc Fru Glu R-infested Tissue:<. Treatment:. TT:.69 R-infested Root herbivory increases root regrowth from the stem R-infested Leaves Stem Roots Figure. Root herbivore attack decreases root invertase activity. Activity of soluble (white, light blue) and insoluble (grey, dark blue) invertases in the leaves (left), stems (middle) and roots (right) of control and root herbivore-infested plants. Starch concentration in different tissues of control and root-infested plants. (c) Sugar concentrations in different tissues of control and root-infested plants. P-values of two-way analysis of variance, including the factor treatment and tissue as well as the interaction term (TT) are shown for each parameter. Asterisks indicate significant factors (P <.5, P <., P <.). Asterisks above or between bars indicate significant differences between treatments within tissues (Holm-Sidak post hoc test: P <.5). Error bars represent standard errors (±SE). To understand whether the observed carbon reallocation patterns and stem morphological changes are associated with changes in the plant s regrowth capacity following root herbivore attack, we excised the belowground root biomass from control and herbivore-infested plants, placed the aboveground portion in water beakers, and measured the number of regrowing stem-borne roots that occurred over a week period. As in the previous experiment, the stem circumference increased more rapidly in D. virgifera infested seedlings (repeated measures anova d d7; interaction treatment time: P <.; Fig. 5a). After root removal, all maize plants rapidly started to regrow new crown roots. After weeks, all plants had replaced their root system with at least five crown roots (Fig. 5b). D. virgifera infested seedlings regrew significantly more roots between and d postinfestation (repeated-measures anova; interaction treatment time: P <.; Fig. 5b). The difference was most pronounced 6 d after root removal, when root-infested plants had more than two additional crown roots compared with control plants. During the regrowth phase, stem thickness was strongly reduced, and no difference between the circumference of D. virgifera and control stems was observed at the end of the experiment (Fig. 5a). Root herbivore-induced root regrowth is independent of a plant s water supply As plant water supply strongly influences the impact of D. virgifera on shoot physiology (Erb et al. ), we conducted an additional regrowth experiment with plants growing under three different water regimes. During repotting, we recorded the extent of D. virgifera damage to the roots under all the different water regimes (two-way anova, F = 7.8, P <.; Fig. 6a). Damage by D. virgifera was John Wiley & Sons Ltd, Plant, Cell and Environment, 7, 6 6

7 Induced root herbivore tolerance 69 Stem circumference (cm) Regrowing crown roots (#) Treatment:.9 Time: <. TT:. 6 7 Time (days) Treatment:.8 Time: <. TT:. Root infested 5 Root removal and regrowth Time (days post-infestation) Figure 5. Root attack increases compensatory root growth. Stem circumference of control and Diabrotica virgifera infested maize plants over the course of infestation (days 7) and at the end of the regrowth phase (day 5). Number of emerging crown roots from control and root-infested plants during the regrowth phase. P-values of repeated-measures analyses of variance, including the factor treatment and time as well as the interaction term (TT) are shown for each parameter. Asterisks indicate significant factors (P <.5, P <., P <.). Asterisks above individual time points indicate significant differences between treatments at this time (Holm-Sidak post hoc test: P <.5). Error bars represent standard errors (±SE). Root damage rating (Oleson s scale) Regrowing crown roots (#) Regrowing crown roots (gdw) Treatment:<. Watering:. TW:. (c) n.d. n.d. n.d. 5 Watering (ml day ) Treatment:. Watering:.75 TW:.88 (e) 5 Watering (ml day ) y =.665x.8 R² =.8 7 d PI Root infested Stem circumference (cm) Post infestation Stem circumference (cm) Regrowing crown roots (gdw) 5.. Treatment:. Watering: <. TW:.58 (d) 5 Watering (ml day ) d PI.8 Treatment:.8 d PI Watering: <..6 TW: Watering (ml day ) 7 d PI Figure 6. Root herbivory increases growth and biomass of stem-borne roots. Root growth of infested and control plants under three different watering regimes was followed after repotting all plants into a root herbivore-free environment to determine whether Diabrotica virgifera induces an increase in regrowth capacity. D. virgifera damage rating using Oleson s scale. Stem circumference 7 d after infestation. (c) Number of regrowing crown roots d post infestation (PI; d after repotting the plants). (d) Total biomass (dry weight) of regrowing roots at the end of the experiment ( d PI). (e) Correlation between stem circumference 7 d PI and the biomass of regrowing roots d PI (values for a linear regression are shown). P-values of two-way analyses of variance, including the factors treatment ( treatment ), watering regime ( watering ) and the interaction term (TW) are depicted for each parameter. Asterisks indicate significant factors (P <.5, P <., P <.). Error bars represent standard errors (±SE). higher in plants that received a medium amount of water per day than in low- and high-watered plants (Holm-Sidak post hoc tests, P <.). Stem circumference was only marginally increased by root herbivory in this experiment (two-way anova, F =.75, P =.; Fig. 6b), but increased with water supply (two-way anova, F = 8.776, P <.; Fig. 6b). After weeks of regrowth in the absence of herbivory, the regrowth of stem-borne crown roots (measured by the total root biomass) in infested plants was significantly higher than in uninfested control plants (two-way anova, F =.885, P <.; Fig. 6c). Furthermore, infested plants regrew more individual crown roots than control plants (two-way anova, F = 7.58, P =.8; Fig. 6d). The watering regime did not John Wiley & Sons Ltd, Plant, Cell and Environment, 7, 6 6 influence this compensatory growth response, but significantly increased the overall biomass accumulation of newly developed roots (two-way anova, F =.8, P <.; Fig. 6d). A strong correlation was observed between the stem circumference measured at 7 d after infestation and the biomass of the regrowing crown roots (Pearson productmoment correlation coefficient:.69; P <.; Fig. 6e). DISCUSSION Our study provides evidence that maize plants respond to root herbivory by allocating more carbon to the stems and by increasing the growth of stem-borne crown roots after the

8 6 C. A. M. Robert et al. attack. The CO radiotracer studies revealed that the whole maize plant reacts dynamically to D. virgifera attack. In the aerial portions, the plant responded by taking up slightly more carbon through CO fixation and by increasing photoassimilate export from source leaves (Fig. ). Below ground, relative competitive sink strength for total plant carbon resources was diminished as evidenced by decreasing root meristem activity for new carbon (as C; Fig. ), and increased shoot-to-root C activity distributions. This decrease in carbon resource allocation to roots was likely promoted by the decreased activity of root invertases (Fig. ) and lead to an accumulation of photoassimilates in the stems. Taken together, our findings add to the growing evidence that plants remobilize resources away from the herbivore damage site and sequester them into inaccessible organs (Babst et al. 5, 8; Schwachtje et al. 6; Gómez et al. ; Hanik et al. a; Xue et al. ). Furthermore, our study demonstrates that plants are able to respond in a spatially congruent fashion by reducing carbon flow to the roots when attacked by belowground herbivores (see also Newingham et al. 7). Based on the observations that D. virgifera attacked plants tended to take in more carbon and mobilized more photoassimilates for export from the source leaves while root import remained constant, we propose that the excess carbon may have been stored in the stems. Indirect evidence for an active role of the stems in aboveground responses to belowground herbivory comes from our morphological observations which document that () the stems of rootattacked plants become thicker and heavier (Fig. ) and () stem thickness is strongly correlated with the regrowth of stem-borne roots (Figs 5 & 6). Grasses are well known to employ their stems as carbohydrate storage organs, for instance to buffer changes in photoassimilate supply (Scofield et al. 9) and to cope with environmental stress (Slewinski ). It is therefore probable that stems are also involved in induced tolerance against root herbivores. How the additional carbohydrates of D. virgifera attacked plants are stored remains to be elucidated. Although sucrose has been suggested as the main storage form of maize stems (Slewinski ), our experiments indicate that neither starch nor soluble sugars served as the additional storage component in the current study, as their levels were not increased in stems of attacked plants (Fig. ). Alternative storage forms that could be investigated in more detail in the future include for instance oligosaccharides (Zhou et al. ) as well as more complex polymers (Halford et al. ). Alternatively, the additional photoassimilates may also be used directly to initiate the growth of new crown root primordia. In the specific case of maize, increased allocation of carbon resources to the stem following D. virgifera attack may serve two purposes. Firstly, D. virgifera larvae may be deprived of essential carbon-based nutrients supplied by the leaves, which may reduce the nutritional value of the roots and prompt the larvae to move away from infested plants. Secondly, maize plants may be able to use the sequestered resources to grow new adventitious roots after the attack is over. Until now, we have little evidence to support the first possibility, as D. virgifera larvae aggregate on host plants and perform better on already attacked root systems (Robert et al. a). On the other hand, our regrowth experiments provide evidence in favour of the second hypothesis, as D. virgifera infested plants regrew more roots than uninfested control plants. The maize root system is composed of several different root types including embryonic primary and seminal roots as well as post-embryonic lateral and crown roots (Hochholdinger & Tuberosa 9). Although embryonic roots are essential during the first weeks of development, the stem-borne crown roots take over all essential functions at later developmental stages (Robert et al. b). This developmental aspect is of significance in the context of the current study, as the regrowing roots of attacked plants were all stem-borne and may therefore have benefited directly from increased carbon allocation to this tissue. Indeed, plants that had to regrow a full-root system displayed a marked reduction in stem thickness, and plants with thicker stems were able to regrow more crown roots. Soil moisture has been shown to be an important determinant of shoot responses of maize-to-root infestation. For instance, well-watered maize plants did not show any change in photosynthetic rates upon infestation in the greenhouse, whereas infested water-stressed plants had lower photosynthetic activity than their non-infested controls (Dunn & Frommelt 998). In a field study, on the other hand, higher photosynthetic activity was measured in the leaves of D. virgifera infested plants (Godfrey et al. 99; Dunn & Frommelt 998), a phenomenon that was interpreted as a component of the compensatory root growth response of D. virgifera tolerant maize lines. Our experiments confirm the notion that maize plants may be able to increase carbon uptake in leaves through CO fixaton following root attack by D. virgifera (Fig. ). The effect of root attack on photosynthesis is likely to be even higher than observed in our assays, as CO assimilation in the gas exchange chambers was above 9% even in control plants, indicating that some plants would have been able to accept even more CO, if available.the fact that some studies report a decrease in photosynthetic activity following D. virgifera attack may be explained by a reduction in water supply from the roots. Especially under waterlimiting conditions, root herbivory increases water stress in maize leaves, which may lead to stomatal closure and reduction of CO assimilation (Erb et al. ). In our radiotracer experiments, plants were grown in Hoagland s fortified agar gel and did not suffer from any water stress following D. virgifera attack. Interestingly, we observed a decrease in root FW, but not root DW following D. virgifera attack, which may point to a potential water loss in attacked roots. However, the increased root regrowth was independent from the plant s water supply (Fig. 6), suggesting that the influence of the larvae on photosynthesis per se is not essential for compensatory growth. It remains to be determined whether the carbon reallocation is equally unresponsive to changes in plant water status. The potential ecological relevance of our findings is twofold. First, our experiments suggest that attacked plants possess the capacity to reprogram the physiology and John Wiley & Sons Ltd, Plant, Cell and Environment, 7, 6 6

9 Induced root herbivore tolerance 6 metabolism of their aerial tissues as a way to reduce the negative impact of root removal. Given that many root feeders seem to be well adapted to the defensive systems of their host plants (Erb et al. ), induced tolerance may be an important alternative strategy for plants to sustain growth in environments that are under high belowground pressure, similarly to what has been documented for aboveground systems (Carmona & Fornoni ). Second, our results demonstrate a potential route by which leaf- and root-feeding insects may influence each other when sharing a host plant. So far, research on the impact of root herbivory on aboveground insects has focused on systemically responsive plant defences (Bezemer & van Dam 5; van Dam et al. 5; Kaplan et al. 8) and water stress-mediated effects on primary metabolites (Gange & Brown 989; Erb et al. 9, ; Johnson et al. ). Induced changes in carbon allocation may also change the host plant s suitability for aboveground feeders, for instance by providing more dietary carbohydrates or, conversely, diluting the available nitrogen and thereby reducing the nutritional value of the tissues. Plant-mediated interactions between root, stem and leaffeeding insects are not only important in an agricultural context, but also from an evolutionary perspective, as they will determine the benefit of herbivore-induced carbon reallocation. Depending on the frequency and severity of attack of different herbivores, root herbivore-induced carbon reallocation may be an advantage or a disadvantage for the plant, and it can be expected that this type of response is only adaptive in ecosystems in which root feeders are the dominant herbivores. To understand the benefits and evolution of root herbivore-induced carbon reallocation, a more detailed understanding of the dynamics in wild systems would be necessary. In this context, it will also be important to evaluate whether the observed responses are herbivore specific. Our current experiments do not allow distinguishing between damage and herbivore-induced effects (Erb et al. ), and further research is necessary to understand the importance of root biomass removal for carbon allocation. The fact that there was no apparent correlation between root damage and root regrowth, however, points to a disproportionate response that goes beyond a potential change in the plants functional equilibrium (Fig. 6). In the long run, studying the specificity of root tolerance responses will likely lead to a better understanding of plant defence strategies and herbivore distribution patterns in terrestrial ecosystems. ACKNOWLEDGMENTS We are grateful to Wade French and Chad Nielson (USDA- ARS-NCARL, Brookings, SD, USA), and Julie Barry (USDA-ARS, University of Missouri, Columbia, MO, USA) who kindly supplied D. virgifera eggs. We thank Lena Kurz for her help with the tolerance measurements. Research activities by C.A.M.R., T.C.J.T. and M.E. were supported by the Swiss National Science Foundation (FN AO- 797; 96) and a Marie Curie Intra European Fellowship (grant no. 77). C.A.M.R. was supported by a travel grant of the Organismal Biology Doctoral Program of the John Wiley & Sons Ltd, Plant, Cell and Environment, 7, 6 6 University of Neuchâtel to conduct experiments at BNL. This article has been authored by Brookhaven Science Associates, LLC under contract number DE-AC-98CH886 with the US Department of Energy, Office of Biological and Environmental Research, which supported R.A.F., B.A.B. and M.J.S. This project was partially funded by the Max Planck Society and the National Centre of Competence in Research (NCCR) Plant Survival, a research programme of the Swiss National Science Foundation. REFERENCES Babst B.A., Ferrieri R.A., Gray D.W., Lerdau M., Schlyer D.J., Schueller M.,... Orians C.M. 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Fresh and dry mass of control and D. virgifera infested shoots and roots from the CO labelling experiment. Figure S. Fresh mass, dry mass and water content of control and D. virgifera infested shoots and roots from the regrowth experiment. John Wiley & Sons Ltd, Plant, Cell and Environment, 7, 6 6