Characterizing plant responses to herbivory: direct induction vs. priming, systemic vs. local responses

Chemical defense responses can result from direct induction, such that the levels are higher after the initial herbivory or cue of herbivory; and/or they can be primed, such that levels of defense are higher or more rapid after subsequent herbivore attack (Frost et al. 2008). In the first experiment below, we sample leaves only after herbivory, allowing us to measure induced defenses elicited by vibration, but not to separate out direct effects vs. priming. In the second experiment, we include a no-herbivory treatment, allowing us to separate out priming from direct effects of chewing vibrations on plant responses. Induced responses can also be local, occurring only in tissues near the site of herbivory, or systemic, occurring over a larger spatial scale within the plant (Kessler and Baldwin 2002). Chewing vibrations are propagated rapidly to other leaves on the plant (Fig. 1a), and thus have the potential to trigger a systemic response. In the experiments below, we examine the potential for both local and systemic effects by sampling the leaf used for playback of chewing vibrations, a same-age leaf on the opposite side of the plant, and the unexpanded leaves in the rosette center.

Fig. 1 a Vibrations produced by a feeding P. rapae caterpillar on A. thaliana, recorded simultaneously (using two laser vibrometers) on the fed-upon leaf and a second leaf on the opposite side of the plant. These leaves correspond to the leaves labeled ‘pbl’ and ‘sl’ in the playback design shown in the next panel. b Sampling design for the experiments. An older leaf was selected for caterpillar recordings and vibrational playback (pbl), to allow attachment of an actuator with minimal effect on the rest of the plant. For the plants that experienced herbivory (all of the plants in experiment 1, and half of the plants in experiment 2), caterpillars were confined in clip cages placed on the playback leaf and a same-age leaf on the opposite side of the plant (sl). The two leaves experiencing herbivory 24 or 48 h after the experimental treatment are marked with an asterisk. The young unexpanded leaves in the rosette center (rc) were also sampled for leaf chemistry, but did not experience herbivory Full size image

Plant growth

A. thaliana Col-0 plants were grown in individual #3 pots (55 × 57 mm) in potting soil (Pro-Mix; Premier Horticulture Inc., Quakertown, PA, USA) supplemented with 1.8 kg of Osmocote™ slow-release fertilizer (The Scotts Company, Marysville, OH) per cubic meter of soil. Plants were grown under metal halide lamps at 24 °C and 62 % relative humidity with a 8:16 h (L:D) 180 µmol m−2 s−1 photoperiod. Vegetative (rosette only) plants used in experiments were 4 weeks post-germination. Two days before the experiments, the plants were transplanted into 50-ml conical plastic centrifuge tubes to maximize the amount of leaf area overhanging the container to use for vibration treatment.

Vibration recordings

To record caterpillar feeding vibrations, we allowed fourth-instar P. rapae caterpillars to feed on a leaf of a potted plant (N = 22 caterpillars and plants) and recorded the vibrations experienced by the fed-upon leaf, and a leaf on the opposite side of the plant (Fig. 1a, b). Chewing vibrations were recorded at 24.5 ± 1 °C with laser Doppler vibrometry (Polytec CLV 1000 and CLV M030 decoder module). To experimentally reproduce the caterpillar feeding vibrations, we used piezoelectric actuators supported under a leaf (Electronic Supplementary Material Fig. 1A) and attached to the leaf using accelerometer mounting wax. Before playback of the vibrations recorded on the fed-upon leaf, we characterized the frequency response of each actuator, then designed a digital filter that compensated for that response (Cocroft 2010). The playback stimuli were then filtered to yield playbacks that closely matched the temporal and spectral properties of the original recordings (Electronic Supplementary Material Fig. 2). We calibrated the amplitude of each playback to match that of the original recording.

We based our playback design on the feeding behavior of P. rapae caterpillars, which spend an average of 100 ± 223 min on a leaf, alternately feeding and resting (Coffman pers. comm.). Our playback stimuli consisted of 10 s of chewing followed by a 10 s pause, repeated for 5 min; there was then a 5 min pause. This basic 10-min pattern was repeated for 120 min to reflect the natural timing of P. rapae feeding activity.

Insect growth and herbivory treatments

P. rapae (L.) were reared at 24 °C on A. thaliana plants grown in pots as described above, and are the progeny of biological stock originally obtained from Carolina Biological and the Jander laboratory (Cornell University, Ithaca, NY). Post-ecdysal fourth instar caterpillars were used for all experiments. Insects were removed from these plants for a maximum of 3 h before use. Caterpillars were placed on an older, outer rosette leaf like those used for vibrational playback, after which most individuals began feeding on the leaf. Laser vibrometry recordings of caterpillar feeding vibrations were made from the fed-upon leaf near the base of the leaf blade. The herbivory treatments were begun 30 min after playback of feeding vibrations. Individual larvae were confined using a clip cage (Electronic Supplementary Material Fig. 1B) to a fully expanded leaf in the rosette, and were allowed to feed until approximately 30 % of the leaf was removed. Leaves experiencing herbivory included the playback leaf and a same-age leaf on the opposite side of the plant (Fig. 1b). The no-herbivore treatment (Experiment #2, below) consisted of empty clip cages on corresponding leaves. Leaves were harvested into liquid N 2 24 and/or 48 h after caterpillar feeding, depending on the experiment.

Defense chemistry

A. thaliana produces three major classes of chemical defenses in greater amounts following insect damage: glucosinolates (GSs: Mewis et al. 2005), the polyphenol anthocyanins (ACs: Ferrieri et al. 2013), and a suite of volatile compounds (Snoeren et al. 2010). Glucosinolate quantification procedures were adapted from previously described protocols (Mewis et al. 2005). Leaves were freeze-dried (2–4 mg DW) before being ground to a fine powder in a Talboys high throughput homogenizer (Troemer, NJ, USA) for extraction. Glucosinolates were extracted three times in 70 % methanol/DI H 2 O at 80 °C for 5 min. Supernatants were pooled and placed in a centrivap until dry. Pellets were re-suspended in 40 µL of 0.4 M barium acetate and 370 µL deionized water to precipitate protein, and desulphated overnight on DEAE Sephadex A-25 in 96-well filter plates. Plates were prepared by vacuum filtration with two 200-µL washes of 6 M imidazole formate followed by three additional washes of DI H 2 O. Crude glucosinolate extracts were added to individual wells and washed twice using sodium acetate buffer solution (pH 4.0). Sulfatase solution (30 µL) was added into to each sample for overnight desulfination at 4 °C. Desulfated glucosinolates were eluted twice in 150 µL of distilled water using a vacuum manifold. Detection and quantification of individual desulfated indolyl and aliphatic glucosinolates was performed using a Waters Alliance 2695 HPLC in tandem with a Waters Acquity TQ detector mass spectrometer, on a C18 RP column using a water/acetonitrile linear gradient. Glucosinolates were monitored by a UV detector at 229 nm and quantified using an internal standard (sinalbin) added prior to extraction. Our HPLC analyses allowed us to quantify the molar concentrations of ten individual glucosinolate compounds, including seven aliphatic glucosinolates [3-methylsulfinylpropyl (3 MSOP), 4-methylsulfinylbutyl (4MSOB), 5-methylsulfinylpentyl (5MSOP), 6-methylsulfinylhexyl (6MSOH), 7-methylsulfinylheptyl (7MSOH), 4-methylthiobutyl (4MTB), and 8-methylsulfinyloctyl (8MSOO)] and 3-indolyl glucosinolates [3-indoyl-methyl-(I3 M), 4-methoxy-3-indolylmethyl-(4MOI3 M), and 1-methoxy-3-indolylmethyl-(1MOI3 M)].

Polyphenols, including anthocyanins, were extracted and quantified as described previously (Ferrieri et al. 2013). Individual leaves were freeze-dried (4–6 mg DW), ground as described above, and extracted and quantified. Phenolics were extracted overnight in 200 μl of 1 % (v/v) HCl in methanol at 4 °C. An additional extraction with 250 μl distilled water and 500 μl chloroform was used to remove chlorophyll. Samples were vortexed and centrifuged for 3 min at 3,000×g. Relative anthocyanin levels in the aqueous phase were determined spectrophotometrically by measuring absorbance at 530 nm. Total flavonoid compounds were also estimated in the same extracts at absorbance 320 nm (Fukumoto and Mazza 2000; Shao et al. 2008). The concentration of total redox-reactive phenolics present in leaf extracts was determined using the Folin-Denis assay, with standard curves developed using chlorogenic and gallic acids, and standards purified from each treatment group (Appel et al. 2001).

Experiment #1

In the first experiment, we played back caterpillar vibrations to naïve A. thaliana plants, using piezoelectric actuators (AE0505D18F actuators and MDT693B and MDR694B controllers, Thorlabs, Inc., Newton, New Jersey, USA). In each replicate (N = 22), we used a different chewing exemplar from the recordings made above. A single replicate included four plants: two plants received the chewing playback, while two received a sham control (silent actuator attached to the leaf). Caterpillar feeding vibrations were played back to plants for 2 h, as described above. Immediately after the playback, we allowed fourth instar P. rapae caterpillars to feed in individual clip cages (Electronic Supplementary Material Fig. 1B) on the vibrated leaf and an age-matched non-vibrated leaf (Fig. 1b) on all plants until approximately 30 % of the leaf area was consumed. At 24 and 48 h later, target and non-target leaves and the central group of unexpanded leaves were removed, flash frozen in liquid nitrogen and stored at −80 °C. Leaves were freeze-dried before analysis of GSs. We evaluated the influence of vibration treatment (chewing vibration vs. no-vibration control), tissue (playback leaf, same-age systemic leaf, unexpanded leaves in center of rosette) and sampling interval (24 vs 48 h) on the concentration of aliphatic and indolyl glucosinolates using a general linear mixed model in SAS v. 9.3 (using PROC GLIMMIX; SAS code provided in Electronic Supplementary Material, Table 1) with a gamma distribution (Bolker et al. 2009). Note that though we sampled the central rosette leaves on fed-upon plants to assess any changes in GS levels, these leaves were not themselves fed upon (it is not feasible to attach clip cages to these unexpanded leaves), and any changes seen in the rosette center would be a response to herbivory on other leaves. The four plants given the vibration treatment at the same time were treated as a block, and the block was included in the model as a random effect. Because we tested two response variables (total aliphatic glucosinolates and total indolyl glucosinolates), resulting p values were adjusted for multiple comparisons using the False Discovery Rate procedure (Benjamini and Hochberg 1995; Garcia 2004).

Experiment #2

The design of this experiment was similar to the previous experiment, but with three differences that allowed us to answer additional questions. First, we asked whether vibrations elicited the production of phenolics, the second major class of defenses in A. thaliana, rather than glucosinolates. Second, we assessed the roles of direct induction (increased defenses in the absence of herbivory) vs. priming (increased defenses in response to herbivory), by adding a ‘no herbivory’ treatment consisting of plants that received the vibration playbacks and clip cages but no caterpillar feeding. Third, we asked whether the response to vibration was specific to chewing vibrations, as opposed to being induced by simply any vibration, a possibility not excluded by the first experiment. To address this question, we included two additional vibration controls: wind-induced vibrations, a common source of vibrational noise in the field; and the vibrational mating song of a leafhopper, chosen because it has a similar frequency spectrum to that of chewing, but a contrasting temporal pattern. Wind-induced vibrations were obtained by directing a small fan at A. thaliana plants of the same size as the experimental plants, and recording the leaf motion using laser vibrometry. Leafhopper recordings were drawn from a library of signals previously recorded by Cocroft.

As above, the amplitude of each chewing playback was matched to that of the original recording. The displacement amplitudes of the wind and leafhopper exemplars in each replicate were matched to that of the corresponding chewing exemplar. Chewing and leafhopper vibrations were played back using piezoelectric actuators, but the wind recordings contained mostly very low frequencies that were better replicated with a magnet and coil extracted from audio speakers (see Cocroft 2010 for a discussion of playback methods). To achieve uniform contact between all actuators and the playback leaf, short lengths of balsa dowel were attached to each transducer (Electronic Supplementary Material Fig. 1A), with the contact between dowel and leaf secured using accelerometer mounting wax. We conducted 18 replicates of the playbacks, each containing eight plants, with two plants per vibration treatment. The vibration treatments included caterpillar chewing, wind-induced vibrations, leafhopper mating song, and a sham with no vibration, as described above. Each replicate used different exemplar recordings (i.e., no recording was used in more than one replicate), and replicate was treated as a block for statistical analysis. The use of two plants for each treatment within a block allowed us to test for both direct induction and priming, by exposing half of the plants in each vibration treatment to caterpillar feeding and half to empty cages. To test for a direct effect, we measured defense chemistry 48 h after herbivory in playback and same-age systemic leaves of plants that experienced the vibration treatments but no herbivory. To isolate priming effects from direct effects, we took the ratio of the responses of the plants in the same replicate that did and did not experience herbivory. Because the matched plants received the same vibration exemplars, any direct effects will appear in both the numerator and denominator of the ratio, canceling out and leaving only the priming effect. Note that, as in Experiment #1, the unexpanded central rosette leaves did not experience herbivory.

We analyzed the data using a general linear mixed model with a gamma distribution, as above, to examine the influence of vibration treatment (chewing, wind, insect song, no vibration control) and tissue (vibrated leaf, non-vibrated same-age leaf, unexpanded leaves in the center of the rosette) on phenolic responses. Replicate (the set of eight plants tested at the same time) was included as a random block effect. Because we measured three response variables (anthocyanins, flavonoids, and phenolic redox activity as measured by the Folin-Denis assay), we used the false discovery rate procedure as described above to adjust the experiment-wide Type I error rate.

Note: the data from this study are provided in the Electronic Supplementary Material 2.