Mechanosensation in Plants by Taylor Y. Paret Submitted to the Graduate

A Thesis

entitled

To Hear Without an Ear: Mechanosensation in Plants

Taylor Y. Paret

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the

Master of Science Degree in

Biology

______

Heidi M. Appel, PhD, Committee Chair

______

Scott A. Heckathorn, PhD, Committee Member

______

Elizabeth S. Haswell, PhD, Committee Member

______

Amanda Bryant-Friedrich, PhD, Dean

College of Graduate Studies

The University of Toledo

May 2019

This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

To Hear Without an Ear: Mechanosensation in Plants.

Taylor Y. Paret

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Master of Science Degree in Biology

The University of Toledo

May 2019

Plants respond to herbivory by increasing the production of chemical defenses.

Early defense signaling depends on the plant’s ability to quickly detect the attack and activate the appropriate signaling cascades. Response cascades begin with the perturbation in plant plasma membrane potential, change in the cells calcium concentration, and production of reactive oxygen species (ROS), eventually leading to an increase in plant chemical defenses. The plant can recognize wounding, insect oral secretions, and insect feeding vibrations to identify the “attacker”, and thereby respond accordingly. Mechanosensitive channel of Small conductance-Like (MSL) channels are located in the plasma membrane, and in chloroplast and mitochondria membranes, of higher plants. MSL channels in plants respond to many of the same stimuli as the

MechanoSensitive (MS) channels in animals. These channels can respond to unique stimuli, including cell wall damage and plant-pathogen interactions. We previously showed that plants respond to vibrations caused by insect feeding by priming the production of chemical defenses that will deter insect feeding. By playing recordings of feeding vibrations produced by the White Cabbage caterpillar, Pieris rapae, back to the

iii plant Arabidopsis thaliana, we can prime the production of chemical defenses in the absence of the insect. In this study, we used A. thaliana wildtype (WT) plants, and msl mutants with nonfunctional MSL genes. Plants received either caterpillar feeding vibrations or a silent sham. Foliar phenolics and pigments were quantified by colorimetric assays and foliar phytohormones were quantified by ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry in tandem (UPLC-ESI-

MS/MS). We hypothesized that one or more of the msl mutants would produce fewer chemical defenses than the co-grown WT when treated with insect feeding vibrations.

Our results showed that plants lacking MSL ion channel were not able to respond to insect feeding vibrations in a similar way as WT plants. More specifically, msl9,10 plants had higher phenolic concentrations only when vibrations were followed by a methyl jasmonate (MeJA) spray (defense elicitor) compared to the control, while msl4,5,6 plants did not respond to the vibration regardless of the water/MeJA spray. Moreover, salicylic acid (SA) defense signaling was absent in msl4,5,6 and msl9,10 mutants compared with

WT plants that displayed a strong increase after the vibration playback. Taken altogether, these results suggest that both msl mutant plants (i) were lacking SA signaling at the beginning of the cascade response, and (ii) had an impaired response to the vibrations.

These results confirm that MSL4, MSL5, and/or MSL6 are needed for an appropriate direct and priming response to insect feeding vibration, and that MSL9 and/or MSL10 are likely to be involved in the direct response only. In addition, in this study, we confirm a direct effect of insect feeding vibrations on SA-signaling and report for the first time a direct, as well as priming effect, on the production of chemical defenses.

Acknowledgements

I would first like to thank Dr. Melanie Body. Without her mentorship and her help

I would not have been able to complete this study in the allotted time. She truly has been a great help and an even better mentor. I would also like to take the time to thank Steve

Murphy, the department’s machinist. He made sure that the growth chambers were operational at all times and was a good man to bounce ideas off of to improve the setup.

And lastly, I would like to thank Dr. Heidi Appel and Dr. Jack Schultz. Without Dr.

Appel, I would not have found a passion for science, and Dr. Schultz for all the time that was spent bounce ideas off of to better understand the science behind this experiment.

Contents

Abstract ...... iii

Acknowledgements ...... 1

Contents ...... 2

List of Tables ...... 4

List of Figures ...... 5

List of Supplements ...... 6

List of Abbreviations ...... 7

List of Symbols ...... 9

Chapter 1 — The Role of MSL Ion Channels in Plant Perception of Insect Feeding

Vibrations ...... 10

1.1 Introduction ...... 10

1.2 Material and Methods ...... 14

1.2.1 Plant Material ...... 14

1.2.1.1 Species and Genotypes ...... 14

1.2.1.2 Growth Conditions ...... 14

1.2.1.3 Vibrational Setup/Playbacks ...... 15

1.2.1.4 Treatments ...... 15

1.2.1.5 Plant Tissue Harvest ...... 17

1.2.2 Chemical Analyses ...... 17

1.2.2.1 Secondary Metabolite and Pigment Assays ...... 17

1.2.2.2 Phytohormones ...... 19

1.2.2.3 Water Content ...... 22

1.2.3 Data Interpretation ...... 23

1.2.4 Statistical Analyses ...... 23

1.3 Results ...... 23

1.3.1 Secondary Metabolites ...... 24

1.3.2 Pigments ...... 26

1.3.3 Phytohormone Signaling ...... 26

1.4 Discussion ...... 29

References ...... 51

Supplements ...... 60

List of Tables

Table 1.1 MSL proteins and their location in Arabidopsis thaliana plants.

Table 1.2. The effect of vibration and MeJA spray on secondary metabolite levels.

Table 1.3. Table containing only statistical analysis for phytohormones that were involved in JA defense, SA defense, and ABA signaling.

List of Figures

Figure 1.1. Experimental setup.

Figure 1.2. Comparisons showing direct, priming, and MeJA effects.

Figure 1.3. Phenolic concentrations (anthocyanins; flavonoids; total phenolics).

Figure 1.4. Photographs of the underside of A. thaliana (wildtype Col-0; double mutant msl9,10; triple mutant msl4,5,6) showing anthocyanin accumulation.

Figure 1.5. Concentrations of phytohormones involved in JA defense signaling.

Figure 1.6. Concentrations of phytohormones involved in SA defense signaling.

Figure 1.7. Abscisic acid concentrations (stress signaling phytohormone).

Figure 1.8. Biosynthetic pathways of phytohormones and secondary metabolites.

List of Supplements

Supplemental Table S1.1. UPLC-ESI-MS/MS parameters. Reaction monitoring conditions for protonated or deprotonated plant hormones ([M − H]+ or [M − H]–).

Supplemental Table S1.2. Water content of A. thaliana.

Supplemental Table S1.3. Secondary metabolite concentrations (anthocyanins; flavonoids; total phenolics) of A. thaliana.

Supplemental Table S1.4. Pigment content (total carotenoids; chlorophylls a and b) of A. thaliana.

Supplemental Table S1.5. Concentrations of growth regulators (gibberellins; cytokinins; auxins), ABA signaling, SA and JA defense signaling of A. thaliana.

List of Abbreviations

ABA ...... 2-cis,4-trans-abscisic acid AUXs ...... auxins CKs ...... cytokinins Col-0 ...... Columbia-0 wildtype D6-ABA ...... [2H6]-cis,trans-abscisic acid D6-iP ...... [2H6]-N6-isopentenyladenine DI ...... deionized GA1 ...... gibberellin 1 GA3 ...... gibberellic acid GA4 ...... gibberellin 4 GA7 ...... gibberellin 7 GAs ...... gibberellins GFP ...... green fluorescent protein H2O ...... water HCl ...... hydrochloric acid IAA ...... indole-3-acetic acid IAA ...... indole-3-butyric acid iP ...... 6-(D2-isopentenyl) adenine iPR ...... 6-(D2-isopentenyl) adenine riboside JA ...... jasmonic acid JA-Ile ...... jasmonoyl-isoleucine LC-MS ...... liquid chromatography mass spectrometry MCA ...... mid1-complienting activity MeJA ...... methyl jasmonate MeSA ...... methyl salicylate MRM ...... multiple reaction monitoring MS ...... mechanosensitive MscS ...... mechanosensitive channels of small conductance MSL ...... mechanosensitive channels of small conductance-like OPDA ...... 13-epi-12-oxo-phytodienoic acid OSCA ...... reduces hyperosmolality-induced calcium increase PC ...... piezo channel 7

SA ...... salicylate T-DNA ...... transfer deoxyribonucleic acid tZ ...... trans-zeatin tZR ...... trans-zeatin riboside UPLC-ESI-MS/MS ... ultrahigh performance liquid chromatography electrospray ionization tandem mass spectrometry UV ...... ultraviolet WT ...... wildtype

List of Symbols

α ...... alpha; statistical threshold (α = 5 %)

Chapter 1

The Role of MSL Ion Channels in Plant Perception of Insect Feeding Vibrations

1.1 Introduction

One of the most common threats to a plant’s survival is herbivory. Plants have co- evolved with herbivorous insects, and thus developed a suite of defenses to help deter herbivore feeding. These defenses can be classified in different (non-exclusive) categories: physical vs. chemical, constitutive vs. inducible, local vs. systemic, and direct vs. indirect.

Physical defenses are morphological traits that act as a mechanical barrier to prevent or deter feeding and/or oviposition, such as leaf toughness (Coley 1983;

Berdegue and Trumble 1996), waxes (Brennan and Weinbaum 2001), thorns/spines

(Schoonhoven et al. 2005), or trichomes (above-ground hairs; Traw and Dawson 2002).

In complement, chemical defenses are secondary metabolites that are produced to prevent future herbivory. These chemicals are non-nutritional compounds designed to inhibit growth, behavior, survival, or even population densities of the attacking species

(Whittaker 1970; Schoonhoven et al. 2005).

The levels of defenses can be classified either as constitutive, the amounts present in healthy/undamaged plant tissue, or as inducible when the density/concentration increases upon herbivory. Sometimes induction can be expressed at a later time, in a physiological process called ‘priming’, that allows for a plant to better respond to subsequent stress in the environment. Following the initial exposure to the stress, priming increases the quantity of chemical defenses produced or accelerates the induced response when a subsequent stress occurs (Frost et al. 2008). Defenses can not only be induced in higher density/concentration at the feeding site (local), but also in undamaged plant tissues away from the site of attack (systemic; Dicke and Baldwin 2010; Heil 2014;

Turlings and Erb 2018).

Chemical defenses can have two modes of action: (i) to deter herbivory (direct effect; i.e. phenolics, tannins, glucosinolates, alkaloids, and terpenes; Duffey and Stout

1996) or (ii) to attract natural enemies of the attacker (indirect effect; i.e. volatile organic compounds and extra-floral nectar; Kost and Heil 2006). The induction of plant chemical defenses is triggered by specific stimuli, such as cell wall damage and elicitors from insect saliva and regurgitation (Giron et al. 2016). We have previously shown that

Arabidopsis thaliana plants that were pre-treated with insect feeding vibrations produced a higher level of defenses than the wildtype (WT) (glucosinolates and anthocyanins) when subsequently fed upon by Pieris rapae caterpillars (priming effect; Appel and

Cocroft 2014). This study also reported that the plant was able to discriminate P. rapae chewing vibrations from other ecologically relevant signals, like wind vibrations or from the vibrations of a leafhopper mating song. However, the underlying mechanism involved in plant perception of feeding vibrations is still unknown.

Plants, like animals, are able to respond to environmental stresses through mechanosensation (e.g. MechanoSensitive (MS) channels in animals and

Mechanosensitive channel of Small conductance-Like (MSL) channels in plants), among other mechanisms. Recognizing these stimuli helps plants to survive and respond adequately to their environment. One of the best-known examples of mechanosensitivity in plants is the Venus Fly Trap. When it “feels” that an insect has landed or crawled in the interior walls of the leaf, the leaf closes (Braam 2005). Stimuli from the surrounding environment perceived by plants can include damage, vibrations, touch, gravity, and osmotic pressure (Jayaraman et al. 2014; Braam and Chehab 2017; Hamment and

Haswell 2017; Landrein and Ingram 2019). The downstream responses to each of these stimuli can vary in which defensive compounds are produced and their quantity.

Mechanosensitive ion channels are involved in the perception of sound, touch, pain, shear force, and osmotic challenge in both invertebrate and vertebrate systems

(Sukharev and Sachs 2012; Katta et al. 2015; Ranade et al. 2015; Martinac and Poole

2018); similarly, MSL ion channels are proposed to provide plants with the ability to recognize mechanical stimuli (Hamilton et al. 2015). The plant reacts to external stimuli through ion channels within the membrane. There are several mechanosensitive ion channels: Mid1-Complimenting Activity (MCA), reduced hyperosmolality-induced

[Ca2+] increase (OSCA), Piezo (PC) and Mechanosensitive channel of Small

12 conductance-Like (MscS-L or MSL) (Basu and Haswell 2017; Hamant and Haswell

2017; Zhang et al. 2019). These channels are transporting cations and anions such as calcium, potassium, magnesium, and barium (Hedrich 2012). These transmembrane channels can be found throughout the cell, and between the interior of a cell and the cell wall.

Like MscS channels, their homologues in plants, MSL channels, are “tension gated” channels, e.g. membrane tension is required to open the channels (Basu and

Haswell 2017). It has been proposed that plasma membrane tension changes can be caused by the environmental stimuli cited above, opening the MSL non-selective ion channels (Haswell 2007). To date, ten MSL ion channels have been identified in A. thaliana, and are located in different organelles, or even different organs, in the plant

(Table 1.1). It has recently been showed that MSL1 is localized in the mitochondria and believed to be involved in signaling in developmental stages (Lee et al. 2016, 2019),

MSL2 and MSL3 help to maintain plastid homeostasis during growth (Veley et al. 2012),

MSL8 has a role in pollen hydration (Hamilton et al. 2015; Hamilton and Haswell 2017), and MSL10 has a role in cell death (Veley et al. 2014). The roles of MSL4, MSL5,

MSL6, MSL7 and MSL9 have not been identified yet.

Here, we hypothesize that the MSL ion channels (located in the plasma membrane and endoplasmic reticulum) are involved in plant perception of insect feeding vibrations.

We test this hypothesis by comparing the level of phenolic defensive compounds and phytohormones involved in defense and stress signaling in msl mutants relative to WT A. thaliana plants. We predict that one or more of the msl mutants will produce fewer

13 chemical defenses than the co-grown WT in response to insect feeding vibrations, and that this will be associated with a change in levels of one or more phytohormones.

1.2 Material and Methods

1.2.1 Plant Material

1.2.1.1 Species and Genotypes

In this experiment, three different genotypes of A. thaliana were used: Col-0 wildtype co-grown (grown together, under the same conditions, with the mutant plants) as a control, a double mutant with two of the MSL ion channels absent (MSL 2x; msl9,10), and a triple mutant with three of the ion channels absent (MSL 3x; msl4,5,6).

Ion channel MSL4, MSL5, and MSL6 are located in the plasma membrane, while MSL9 and MSL10 are located in the plasma membrane and endoplasmic reticulum (Table 1.1).

The msl mutant and WT co-grown genotypes were provided by the Haswell lab

(Washington University in St. Louis, St. Louis, MO, USA) and created using a reverse genetics T-DNA (transfer DNA) insertional mutagenesis, as described in Haswell et al.

(2008).

1.2.1.2 Growth Conditions

A. thaliana (Brassicaceae) Col-0 WT and msl mutant plants were grown in individual 2-inch circular pots (#3, 55 x 57 mm) in Pro-Mix potting soil (Premier

Horticulture Inc., Quakertown, PA, USA) and enriched with 1.635 kg of Osmocote™ slow-release fertilizer (The Scotts Company, Marysville, OH, USA) per cubic meter of soil. The plants were grown in Percival Intellus Environmental Controller-AR66L growth 14 chambers (Percival Scientific, Perry, IA, USA) with a photoperiod of 8:16 h

(Light:Dark), under a light intensity of 180 µmol.m-2.s-1, temperature of 22 °C, and relative humidity of 62 %.

1.2.1.3 Vibrational Setup/Playbacks

Vibrations from third to fourth instar P. rapae caterpillars (Figure 1.1C) were previously recorded in the Cocroft Lab (University of Missouri, Columbia, MO, USA) using a Polytec CLV 1000 with CLV MO30 Decoder laser vibrometer (Polytec, Dexter,

MI, USA) (Figure 1.1A). Recordings were cropped into 30 second clips with a one second fade in and one second fade out, using Audacity® (free audio waveform editor).

Filtering the vibrations using a GUI created by the Cocroft Lab on MATLAB

(MathWorks, Natick, MA, USA) allowed for recordings to be “scrubbed” of background noise for an output of higher fidelity to the original signal emitted during caterpillar feeding (Figure 1.1B). The program Reaper (Cockos, New York City, NY, USA) looped the vibrations in the following sequence: 30 seconds feeding vibrations – 10 second silence – 30 seconds of feeding vibrations – 10 second silence, mimicking P. rapae feeding behavior (Appel and Cocroft 2014). Looped recordings were played through a

Tascam Celesonic US20x20 interface (TEAC America Inc., Montebello, CA, USA) and a

Niles SI-1230 Systems amplifier (CORE Brands, Petaluma, CA, USA). The feeding vibrations were then played through 2-inch, 8 ohms, 5-watt speakers (DongGuan,

GuanDong, China) which have been modified with plastic cones and graphite rods

(CynKen Logistics, Shenzhenshi, GuanDong, China) (Figure 1.1E).

1.2.1.4 Treatments

Experiments were conducted on healthy, 5-6 weeks in age, and pre-reproductive

A. thaliana plants. Before each experiment, leaves were numbered in order from youngest to oldest (Figure 1.1D). The smallest leaf over the size of 6 mm inside the rosette was designated as leaf number 1. The day of treatment, a medium aged leaf

(typically leaf #7 or leaf #8) was chosen as the target leaf to receive the vibration playback. Vibration playback occurred in isolation boxes containing five plants of the same treatment/genotype to avoid plant-to-plant communication between different treatments/genotypes via volatile organic compound release (Figure 1.1F).

Vibrations – Each target leaf was set in contact with a graphite rod connected to the 2” speaker using orthodontic wax (Sonic Dental Supply, Bradenton, FL, USA)

(Figure 1.1E). Half of plants received the P. rapae feeding vibrations, while the other half received a silent sham. After 2 hours, plants were removed from the play back system and sprayed with either MeJA or deionized water (DI H2O).

Methyl Jasmonate – Methyl Jasmonate (MeJA) and other Jasmonic Acid (JA) derivatives are referred to as “wounding hormones” and are used to elicit chemical defense responses (Yang et al. 2015; Zhang et al. 2015; Fedderwitz et al. 2016; Lundborg et al. 2016; Tianzi et al. 2018). Plants received an atomized spray (Flo-Masterä atomizer) of either 14 µM of MeJA (Sigma-Aldrich, St. Louis, MO, USA) or DI water

(used as a control). Our previous studies (Appel et al. personal communication) demonstrated that this concentration of MeJA was high enough to induce phenolic response and still permit detection of an induced response from feeding vibrations. The spray was applied for 10 seconds at a 45-degree angle, and then plants were rotated 180 degrees for another 10 second application at a 45-degree angle.

1.2.1.5 Plant Tissue Harvest

After treatments were applied, plants were incubated for either three hours (Body et al. in prep) for phytohormone analysis at conditions listed above, or for 48 hours

(Ferrieri et al. 2015) for phenolic analysis at conditions listed above except the photoperiod was changed to 16:8 h (Light:Dark) to promote the production of anthocyanins (Figure 1.1G). Incubation occurred in isolated growth chambers for each treatment to avoid plant-to-plant communication between different treatments via volatile organic compound release. After the 3-hour or 48-hour incubation, there was no water or

MeJA spray residue left on the plants. Target leaves were harvested by cutting at the base of the petiole. For phytohormones, fresh weight of each sample (pool of five target leaves) was recorded at time of harvest and flash frozen in liquid nitrogen in 2 mL

Eppendorf tubes (Fisher Scientific, Waltham, MA, USA), before being ground into a fine powder using a Biospec MBB – 96 Mini-Beadbeater (Biospec Products Inc., Bartlesville,

OK, USA) and a frozen aluminum tube rack for 1 minute at 3,800 r.p.m. For phenolics, each sample (one target leaf) was immediately weighed (fresh mass), and flash frozen in liquid nitrogen in a 1.2 mL cluster tube (Fisher Scientific, Waltham, MA, USA), before being freeze-dried in a lyophilizer (Genesis 25 SQ Super ES; VirTis – SP Scientific,

Gardiner, NY, USA) for 15 hours under the following conditions: 10 hours at -40 °C, 3 hours at -20 °C, 2 hours at 0 °C, and then held at 20 °C. The pressure was kept below 100 mTorr. The samples were then weighed again (dry mass) and ground into a fine powder for 5 minutes at 3,800 r.p.m, as described above

1.2.2 Chemical Analyses

1.2.2.1 Secondary Metabolite and Pigment Assays

The extraction protocol was adapted from Mancinelli et al. (1988), Fukumoto and

Mazza (2000), Shao et al. (2008), and Choi et al. (2009).

Secondary metabolites were extracted overnight in 200 µL of 1 % (v/v) hydrochloric acid (HCl) in methanol at 4 °C. The samples were inverted manually 100 times over a period of 45 seconds to ensure that all powder was saturated with the extraction solution. After 24 hours, samples were centrifuged for 2 minutes at 2,000 r.p.m. Five hundred µL of chloroform and 350 µL of DI water were added to each sample. Samples were then manually inverted 100 times for 45 seconds to separate the pigments from the chemical defenses. Samples then were centrifuged for 2 minutes at

2,000 r.p.m. Two layers formed, the top aqueous (clear/pink) layer contained the phenolics (among other compounds) and the bottom (cloudy/green) layer containing the chloroform with carotenoids and chlorophylls (Figure 1.1I).

To measure structural groups of phenolics, 130 µL of the top layer of each sample were transferred into a 96 well full-area UV microplate (Fisher Scientific, Waltham, MA,

USA) in three technical replicates. On each plate, two sets of standards were placed: chlorogenic acid for total phenolics and flavonoids (Ark Pharm Inc; Fisher Scientific,

Waltham, MA, USA), cyanidin-3-o-glucoside chloride (Targetmol; Fisher Scientific,

Waltham, MA, USA) for anthocyanin.

To measure carotenoids and chlorophylls, 130 µL of the bottom layer were transferred to a 96-well full-area plate. On each plate, standards were placed: total chlorophylls for chlorophylls a and b (TCI America; Fisher Scientific, Waltham, MA,

USA) and β-carotene for carotenoids (Alfa Aesar; Fisher Scientific, Waltham, MA,

USA).

The microplates were spectrophotometrically read (SynergyHT; BioTech,

Hercules, CA, USA) at 530 nm for anthocyanins, 280 nm for flavonoids, 320 nm for total phenolics, 490 nm for carotenoids, and 670 nm for chlorophylls. Calibrations curves for both chlorogenic acid and cyanidin-3-o-glucoside chloride were prepared using stock solutions at 0.1 mg/mL in DI water, and a serial dilution at: 0, 14, 28, 42, 56, 70, 84, and

98 µg/mL. Calibration curves for carotenoids were prepared using stock solutions at 0.5 mg/mL in 1 % (v/v) hydrochloric acid (HCl) in methanol, and a serial dilution at: 0, 0.07,

0.14, 0.21, 0.28, 0.35, 0.42, and 0.49 mg/mL. Calibration curves for chlorophylls were prepared using stock solutions at 0.3 mg/mL in DI water, and a serial dilution at: 0, 0.42

0.084, 0.126, 0.168, 0.210, 0.252, and 0.294 mg/mL.

1.2.2.2 Phytohormones

We chose to study phytohormones involved in growth and development (GAs, gibberellins; CKs, cytokinins; AUXs, auxins), source-sink relationships (CKs), response to stress (ABA, abscisic acid), and defenses (SA, salicylate; JA, jasmonates). To increase our ability to interpret results, we included several alternative forms of GAs, CKs, AUXs,

SAs, and JAs.

Phytohormone standards – gibberellin 1 (GA1), gibberellin 4 (GA4), gibberellin 7

(GA7), 6-(Δ2-isopentenyl) adenine (iP), 6-(Δ2-isopentenyl) adenine riboside (iPR), trans-zeatin (tZ), trans-zeatin riboside (tZR), indole-3-acetic acid (IAA), 2-cis,4-trans- abscisic acid (ABA), jasmonic acid (JA), 13-epi-12-oxo-phytodienoic acid (OPDA; JA precursor), jasmonoyl-isoleucine (JA-Ile; bioactive JA derivative), methyl jasmonate

(MeJA) – and stable isotope-labeled compounds – [2H6]-cis,trans-abscisic acid (D6-

ABA) and [2H6]-N6-isopentenyladenine (D6-iP) – were purchased from OlChemIm Ltd.

(Olomouc, Czech Republic). Gibberellic acid (GA3), indole-3-butanoic acid (IBA), salicylic acid (SA), and methyl salicylate (MeSA) were purchased from Sigma-Aldrich

(St. Louis, MO, USA). Double distilled (MilliQ) water was used throughout the experiment.

The protocol for phytohormone analysis by ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry in tandem (UPLC-ESI-

MS/MS) was as described in Body et al. (2019), extractions were performed at the

University of Toledo, while the samples were processed at the Charles W. Gehrke

Proteomics Center (University of Missouri, Columbia, MO, USA).

Fifty µL of the working solution of internal standards (500 ppb) and 2 mL of extraction solvent, 2-propanol:H2O:concentrated HCl (2:1:0.002, vol:vol:vol) was added to each 15 mL tube containing the frozen material. Samples were shaken with a tube rotator (J.A.G. Industries Inc., Bastimore, MD, USA) at 80 r.p.m. for 30 minutes at 4 °C before adding 1 mL dichloromethane. Tubes were shaken again for 30 minutes at 4 °C and centrifuged at 4 °C at 13,000 x g for 30 minutes. After centrifugation, two phases were formed; plant debris was located between the two layers (Figure 1.1H). About 2 mL of the solvent from the lower phase were transferred and filtered through a 0.2 µL syringe filter (Whatman plc, Maidstone, United Kingdom) into a 2 mL Eppendorf tube. The solvent was evaporated under a gentle nitrogen gas flow; meanwhile, a second extraction was performed by adding 1 mL dichloromethane to each sample, shaking them for 30 minutes at 4 °C and centrifuging them at 4 °C at 13,000 x g for 20 minutes. About 1.5 mL of the solvent from the lower phase were transferred and filtered at 0.2 µL into the

20 same 2 mL Eppendorf tube. The solvent mixture was concentrated (not completely dry) under a gentle nitrogen gas flow. Samples were then re-dissolved in 500 µL of methanol.

Samples were then ran through a 96-well solid phase extraction (SPE) plate. The plate was conditioned with 250 µL of 100 % methanol, 250 µL of 50 % methanol and 0.1

% formic acid, and finally 750 µL of 0.1 % formic acid. Each conditioning solvent was pulled through the membrane under a gentle vacuum until each well was fully drained.

Once the plate was conditioned 500 µL of the sample was added to each well with 500

µL of 100 % methanol (the dilution allowed the extract to slowly run through the membrane for a better recovery rate) and vacuumed through the membrane. The membranes were washed with 2 mL of 0.1 % formic acid. To elute the samples, a 96-well collection microplate was placed under the SPE plate, 50 µL of 100 % methanol was added to each well, and centrifuged for 30 seconds at 3,500 x g. The samples were transferred from the 96-well collection plate into a 150 µL insert in a 2 mL LCMS- certified Amber glass vials (Waters, Milford, MA, USA).

Ten µL of sample solution were injected twice (for either positive or negative ion mode; Body et al. 2019) into an UPLC-ESI-MS/MS for quantification of all phytohormones. The phytohormones were separated by a reverse-phase BEH C18 UPLC column (Waters® 1.7 µm C18 130 Å, UPLC column 50 mm x 2.1 mm; Waters, Milford,

MA, USA) on a Waters Acquity H-class ultrahigh-performance liquid chromatography

(UPLC) (Milford, MA, USA) system coupled with a UV detector (Waters 996

Photodiode Array Detector) and a Waters Acquity TQ triple quadrupole mass spectrometer (MS/MS) (Waters TQ Detector, Acquity Ultra Performance LC). The binary solvent system used for phytohormone separation was 0.3 mM ammonium

21 formate in MilliQ water (negative-ion mode) or 0.1 % formic acid in MilliQ water

(positive-ion mode) (mobile phase A) and 100 % methanol (negative-ion mode) or 0.1 % formic acid in acetonitrile (positive-ion mode) (mobile phase B) set as follows for a 5- minute run: initial conditions were 2 % B, hold at 2 % B for 0.1 minute, gradient of 2-40

% B over 1 minute, ramp 40-98 % B over 1.5 minutes, hold at 98 % B for 1 minute, rapid ramp (0.2 min) to and hold at (1.3 minutes) initial conditions. The samples were cooled to

10 °C in the autosampler, the oven (holding UPLC column) temperature was set at 30 °C and the solvent flow rate at 0.4 mL.min-1. For each phytohormone, the full spectrum of the protonated [M + H]+ or deprotonated [M − H]- molecular ion and its product ions was generated; the predominant fragmented ion was selected as the product ion for quantification. The ion source in the MS/MS system was electrospray ionization (ESI) operated in the positive or negative ion mode (Body et al. 2019) with capillary voltage of

1.5 kV. The ionization sources were programmed at 150 °C and the desolvation temperature was programmed at 450 °C. The MS/MS system was operated in the multiple reaction monitoring (MRM) mode with the optimized collision energy. The ionization energy, MRM transition ions (precursor and product ions), capillary and cone voltage, desolvation gas flow, and collision energy were optimized by Waters

IntelliStartTM optimization software package. The optimized parameters for the method are reported Supplemental Table S1.1.

1.2.2.3 Water Content

Samples used for phenolic analysis were weighed at harvest and after the freeze- drying process. The weight difference between fresh and dry mass allowed us to calculate the water content (Supplemental Table S1.2) of all samples from all genotypes and

22 treatments. Water content was similar for all conditions allowing us to directly compare the phytohormone concentrations that were determined on fresh tissue.

1.2.3 Data Interpretation

The comparison between sham + water and vibration + water treatment show the direct effect of insect feeding vibrations, while the comparison between sham + MeJA and vibration + MeJA treatment show the priming effect (Figure 1.2).

The comparison between sham + water and sham + MeJA, or between vibration + water and vibration + MeJA, show the effect of the MeJA spray (Figure 1.2).

1.2.4 Statistical Analyses

All statistical analyses were conducted in SAS version 9.4 (SAS Institute

Software Company, Cary, NC, USA). Secondary metabolite concentrations had a normal distribution, while phytohormone concentrations had a gamma distribution. For consistency, both were analyzed using a general linear mixed model (GLM, PROC

GLIMMIX). The model included ‘vibration’, ‘MeJA’, ‘genotype’, as fixed effects, and

‘round’ as a random effect. All p-values (p) were adjusted using the false discovery rate

(FDR) correction procedure of Benjamini and Hochberg (1995) for the use of multiple variables.

1.3 Results

In this study, we recorded changes in A. thaliana secondary metabolite and phytohormone concentrations in response to insect feeding vibrations and MeJA spray in wildtype (WT), double mutant (msl9,10), and triple mutant (msl4,5,6) plants. We quantified the chemical defenses (anthocyanins, flavonoids, total phenolics) and pigments 23

(carotenoids, chlorophylls) by colorimetric assays (N = 20 per genotype per treatment).

We also monitored 17 phytohormones (N = 15 per genotype per treatment) belonging to different processes: defense signaling (JA-pathway, SA-pathway), stress signaling

(abscisic acid), and growth regulators (gibberellins, cytokinins, auxins), by UPLC-ESI-

MS/MS.

1.3.1 Secondary Metabolites

Results for the anthocyanins, flavonoids, and total phenolics assays are presented in Figure 1.3 and GLM statistical analyses are presented in Table 1.2 (concentrations are available in Supplemental Table S1.3).

In the WT plants, vibrated plants produced up to 32 % more anthocyanins than control plants, irrespective of subsequent treatment (GLM, p ≤ 0.01). The double mutant, msl9,10, did not respond to the vibrations with the water treatment (GLM, p = 0.228); however, a strong effect (+38 %) was observed when the vibrations were followed by

MeJA spray (GLM, p ≤ 0.001). The triple mutant, msl4,5,6, did not respond to the vibrations irrespective of the subsequent treatment (GLM, p > 0.05 for water and MeJA).

Both msl mutants had constitutively higher levels of anthocyanins than the WT plants

(Figures 1.3 and 1.4). In WT plants, MeJA spray led to an increase of anthocyanin concentrations by 43 %, regardless of the prior sham/vibration treatment (GLM, p ≤ 0.05 for sham and for vibration). In msl9,10 plants, anthocyanin concentrations were higher after MeJA spray only if the plants received the vibration treatment (GLM, p > 0.05 for sham, p ≤ 0.05 for vibration). On the contrary, in msl4,5,6 plants, anthocyanin concentrations were statistically higher after MeJA spray only if the plants received the sham treatment (GLM, p ≤ 0.01 for sham, p = 0.072 for vibration). 24

WT plants produced up to 46 % more flavonoids when vibrated and then treated with MeJA than plants treated with sham vibrations and/or water (GLM, p ≤ 0.01). The double mutant only responded when vibrations and MeJA were combined (GLM, p ≤

0.05). The triple mutant did not respond to vibrations with either water or MeJA spray

(GLM, p > 0.05 for water and MeJA). Both msl mutants had constitutively higher concentrations of flavonoids than the WT plants (Figure 1.3). In WT and msl9,10 plants, flavonoid concentrations were higher after MeJA spray only if the plants received the vibration treatment (GLM, p ≤ 0.01 for WT, p ≤ 0.05 for msl9,10). On the contrary, in msl4,5,6 plants, flavonoid concentrations were statistically higher after MeJA spray only if the plants received the sham treatment (GLM, p ≤ 0.05 for sham, p = 0.112 for vibration).

WT plants produced up to 30 % more total phenolics when vibration treatment was followed by either water spray, compared with sham + water (GLM, p ≤ 0.01), or

MeJA spray, compared with sham + MeJA (GLM, p ≤ 0.001). Double mutant plants did not increase their defenses when water spray followed the vibrations, compared with sham + water treatment (GLM, p = 0.102), but did produce 15 % more total phenolics when vibrated and sprayed with MeJA, compared to sham + MeJA (GLM, p = 0.060).

The triple mutant did not respond to vibrations regardless of the following treatment

(GLM, p > 0.05 for water and MeJA). Both msl mutants had constitutively higher concentrations of total phenolics than the WT plants (Figure 1.3). In WT plants, MeJA spray led to an increase in total phenolics only if the plants received the vibration treatment (GLM, p > 0.05 for sham, p ≤ 0.05 for vibration). None of the msl mutant

25 plants displayed a change in concentration of total phenolics after MeJA spray, regardless of the vibration treatment (GLM, p > 0.05 for sham and vibration).

1.3.2 Pigments

Results for the assays for carotenoids and chlorophylls are presented in

Supplemental Table S1.4. The pigment concentrations were not impacted by either vibration, treatment, or genotype (GLM, p > 0.05).

1.3.3 Phytohormone Signaling

JA Defense Signaling — Results for the JA-pathways are presented in Figure 1.5 and GLM statistical analyses are presented in Table 1.3 (concentrations are available in

Supplemental Table S1.5).

OPDA (JA precursor) concentrations were not influenced by vibration, treatment, or genotype (GLM, p > 0.05).

JA concentrations were not affected by the vibration treatment, regardless of the water/MeJA treatment or genotype (GLM, p > 0.05). JA concentrations increased by 149

% in WT plants treated with silent sham + MeJA (GLM, p ≤ 0.001), and by 63 % in plants treated with insect feeding vibration + MeJA (GLM, p ≤ 0.001). The double mutant, msl9,10, also showed a large increase (up to 200 %) in JA when sprayed with

MeJA regardless of the sham/vibration treatment (GLM, p ≤ 0.001 for sham and vibration). The triple mutant, msl4,5,6, followed the same pattern as the double mutant with an increased concentration of JA up to 172 % depending on if the plant received silent sham or insect feeding vibrations (GLM, p ≤ 0.001 for sham and vibration).

Similarly to JA, JA-Ile concentrations were not affected by the insect feeding vibration treatment, regardless of the water/MeJA treatment or genotype (GLM, p >

0.05). WT plants sprayed with MeJA had an increase in JA-Ile concentrations of 63 % for silent sham and 84 % for the insect feeding vibrations (GLM, p ≤ 0.05 for sham and vibration). Double mutant plants, msl9,10, had a higher JA-Ile concentration when the plants treated with silent sham (48 %; GLM, p = 0.053) or insect feeding vibrations (56

%; GLM, p ≤ 0.01) when followed by a MeJA spray, compared with water control. Triple mutant plants, msl4,5,6, also had a higher concentration of JA-Ile of 68 % when treated with insect feeding vibrations and sprayed with MeJA compared to silent sham and water spray (GLM, p ≤ 0.05). However, when plants received only one of the treatments, i.e. vibration + water or sham + MeJA, JA-Ile concentrations increased only slightly, by 29

% and 49 %, respectively, compared to plants that received sham vibrations and water spray (GLM, p > 0.05).

MeJA concentrations did not change with vibration, treatment, or genotype

(GLM, p > 0.05)

SA Defense Signaling — Results for phytohormones in the SA-pathway are presented in Figure 1.6 and GLM statistical analyses are presented in Table 1.3

(concentrations are available in Supplemental Table S1.5).

WT plants that received insect feeding vibrations had higher SA concentrations,

68 % higher when the subsequent treatment was water (GLM, p ≤ 0.01), and 40 % higher when the subsequent treatment was MeJA (GLM, p ≤ 0.05). MeJA spray did not affect

SA concentrations regardless of which vibration treatment the plants received (GLM, p =

0.078 for sham, 0.258 for vibration). The double mutant, msl9,10, and the triple mutant msl4,5,6, did not respond to either vibrations or MeJA (GLM, p > 0.05).

Insect feeding vibrations and MeJA spray did not influence MeSA concentrations for the WT plants or the triple mutant, msl4,5,6 (GLM, p > 0.05). However, in the double mutant, msl9,10, insect feeding vibrations led to a decrease of MeSA concentrations of up to 10 % when the subsequent treatment was MeJA spray when compared to silent sham and water spray (GLM, p ≤ 0.05); however, no effect was observed when the subsequent treatment to vibration was water spray (GLM, p > 0.05).

ABA Signaling — Results for ABA are presented in Figure 1.7 and GLM statistical analyses are presented in Table 1.3 (concentrations are available in

Supplemental Table S1.5).

In WT plants, the only significant change in ABA concentration was observed in plants that received both insect feeding vibrations and MeJA spray. The combined treatments led to an increase of 29 % when compared to the insect feeding vibration and water control (GLM, p ≤ 0.01). In the double mutant, msl9,10, plant that were vibrated had a higher ABA concentration (22 %) when treated with MeJA compared with water

(GLM, p ≤ 0.01). In the triple mutant, msl4,5,6, the only statistically significant effect of treatment was observed between plants that received the silent sham and water control treatments and plants that received insect feeding vibrations and MeJA treatments, with an increase of 20 % of ABA concentrations (GLM, p ≤ 0.05).

Growth Regulators — Results for the gibberellins, cytokinins, and auxins are presented in Supplemental Table S1.5. Concentrations of the growth regulators were unaffected by vibrations and/or MeJA treatments, in all genotypes (GLM, p > 0.05).

1.4 Discussion

A. thaliana WT plants had higher foliar levels of chemical defenses after MeJA spray, confirming that MeJA acts as a “wounding hormone” to elicit chemical defense responses (Yang et al. 2015; Zhang et al. 2015; Fedderwitz et al. 2016; Lundborg et al.

2016; Tianzi et al. 2018). Anthocyanin, flavonoid and total phenolic concentrations were also higher in WT plants that were treated with insect feeding vibrations, regardless of the subsequent water/MeJA treatment (Figure 1.3). This is the first report of both a direct and priming effect on the production of chemical defenses in response to insect feeding vibrations. Detection of a direct response was probably due to improvements in the experimental design with the addition of isolation playback chambers to prevent plants from experiencing the volatiles released by plants receiving treatments different from their own.

We hypothesized that plant mechanosensitive ion channels may be involved in the perception of these vibrations and tested the role of two sets of mechanosensitive ion channels using A. thaliana mutants msl4,5,6 and msl9,10. The msl9,10 plants had no direct response to vibrations but retained the priming response, indicating that the MSL9 and/ or MSL10 ion channels are necessary for a priming response to feeding vibrations, but not for a direct response. The msl4,5,6 plants had neither direct or priming responses to vibrations, indicating that the MSL4, MSL5, and/or MSL6 ion channels are necessary

29 for both direct and priming responses to feeding vibrations. Given the predicted cellular locations of these mechanosensitive channels (Table 1.1), these results suggest that both the plasma membrane and/or the endoplasmic reticulum (ER) could be involved in direct responses to vibrations and the ER may not be involved in priming responses to vibration.

To explore the signaling pathways that may be involved in plant responses to vibrations, we measured phytohormone levels 5 hours after their 2-hour vibration treatment began. In WT A. thaliana, none of the common phytohormone components of the JA-pathway (OPDA, JA, JA-Ile, MeJA) responded to insect feeding vibrations.

However, MeJA sprayed onto the plants after the sham or vibration playback led to a strong increase in JA and JA-Ile concentrations, and no change in MeJA concentrations

(Figure 1.5). Taken together, these results suggest that the plant was able to absorb and assimilate the MeJA that was sprayed onto the leaves to catalyze the downstream synthesis of the JA-pathway (Figure 1.8). Both mutant genotypes showed a pattern similar to that observed in the WT plants, with no JA-response to insect feeding vibrations, but a response to the MeJA spray. The JA pathway has been implicated in plant responses to touch and to single tones (Chehab et al. 2012; Ghosh et al. 2016) and we have evidence that the JA pathway is upregulated by feeding vibrations (Body et al. in prep); however, the sampling time (5 hours) in this experiment may have been too early to see an accumulation of JA pathway metabolites.

Alternatively, the JA pathway may have been suppressed by an increase in SA.

Insect feeding vibrations led to higher SA concentrations in WT plants, regardless of the water/MeJA treatment (Figure 1.6). This result is consistent with the previously reported

30 antagonistic crosstalk between the JA- and SA-pathways in A. thaliana (Thaler et al.

2012). No SA response to either vibrations or MeJA was observed in either of the msl mutant genotypes tested in this study. This suggests that in the absence of MSL4, MSL5,

MSL6, MSL9, and/or MSL10 ion channels, A. thaliana can no longer perceive and induce a SA-response to the insect feeding vibrations. Results for MeSA are quite different with no response to vibration or MeJA spray in WT and msl4,5,6 mutant plants, and a decrease of MeSA concentrations in msl9,10 plants that received the vibration +

MeJA treatment compared with sham + MeJA. It seems that this difference is due more to an elevated MeSA level in plants treated with sham + MeJA than a decrease in plants treated with vibration + MeJA. We are currently unable to explain this change.

In msl4,5,6 mutant plants, the lack of SA-signaling in response to the vibrations is consistent with the absence of changes in anthocyanin, flavonoid, and total phenolic concentrations as they are all downstream of the shikimate pathway (Figure 1.8).

However, the conclusion is not as clear for msl9,10 mutants which did not display any change in SA-signaling but still produced more anthocyanins, flavonoids, and total phenolics when treated with vibration + MeJA. Two, non-exclusive explanations are possible: (i) another phytohormone (such as ABA) activated the production of such chemical defenses, or (ii) the increase in phenolics in msl9,10 mutants could be a response to plant-to-plant communication via volatile organic compound released by WT plants because plants that received the same sham/vibration and water/MeJA treatment were incubated in the same growth chamber regardless of their genotype. Indeed, b- ionone was increased in WT plants that received 24-hour of insect feeding vibrations

(Body et al. submitted), and b-ionone and ABA share the same precursor, b-carotene in

31 the tetraterpenoid biosynthesis pathway (Figure 1.8). Nonetheless, MSL9 and/or MSL10 can still be involved to some extent in plant perception of insect feeding vibrations as msl9,10 did not display the phytohormone and chemical profiles similar to those of WT plants. Another study reported that MSL9 and/or MSL10, at least in the roots, are responsible for growth, stress responses, ROS sensing, hormone action, gravitropic responses, and heat perception (Demidchik et al. 2018).

Abscisic acid concentrations were higher in WT plants that received vibration +

MeJA (Figure 1.7) compared to plants receiving sham or vibration followed by a water spray. Average ABA levels were also higher in mutants, although the difference with WT plants was not statistically significant). If the absence of any of the MSL ion channels we tested has any impact on the ABA signaling, it is only in its amplitude as the trend observed in WT plants was still present in the mutants.

In a similar study, A. thaliana WT plants treated with insect feeding vibrations for

24 hours had significantly lower phytohormone concentrations than plants that received the sham vibrations, except for JA that tended to be higher (Body et al. submitted). This pattern resembles what has been reported in plant responses to cold (Wisniewski et al.

2018). The difference in response between this study (sampled at t = 5 hours: 2-hour vibration playback + 3-hour incubation) and Body et al. (submitted; sampled at t = 24 hours) suggests that the plant response to insect feeding vibrations is dynamic over time.

The reported negative crosstalk between jasmonate and salicylate is most likely time driven as well. We suggest that SA may be the early start of the defensive cascade that protects plants from both biotic and abiotic stresses (Thaler et al. 2012). Plant response to insect feeding vibrations starts with high concentrations of SA (our study), and over time,

SA concentrations begin to drop while JA concentrations begin to increase (Body et al. submitted). We have seen similar temporal patterns in plant responses to feeding by another caterpillar, Spodoptera exigua (Appel et al. 2014).

Zou et al. (2016) reported that a gain-of-function mutation (rea1 mutant) in the C- terminus of MSL10 of A. thaliana triggered cell death and the wound-induced early signal transduction pathway and positive feedback synthesis of JA. Providing evidence that

MSL10 may regulate additional pathways (i.e. auxins) involved in the early events of biotic stress signaling. In their study, a hyper-accumulation of JA was recorded 8 hours after wounding; therefore, a longer incubation time in our study may have revealed a different response of msl9,10 to insect feeding vibrations after JA-pathway activation compared with WT and msl4,5,6 mutant plants.

Differences between treatments in the anthocyanin and flavonoid concentrations we measured were not always reflected in similar differences in the total phenolic concentrations. Since these chemical defenses were quantified using colorimetric assays of structurally related groups of phenolics and do not allow for the determination of individual phenolic compounds, the concentrations of other compounds that we did not monitor could have decreased under the same treatment and compensated for the observed increase in anthocyanins and flavonoids. A detailed characterization of individual phenolics could reveal trade-offs between compound biosynthesis, degradation, or activation of different routes.

What can we conclude from this and other experiments on how plant responses to feeding vibrations are organized? We have previously shown that A. thaliana responds to insect feeding vibration priming by increasing the concentrations of anthocyanins and

33 aliphatic glucosinolates (Appel and Cocroft 2014). Here, we report increased concentrations of SA and phenolics. Body et al. (submitted) reported a direct effect of insect feeding vibrations on phytohormone concentrations ([gibberellins] GA1, GA3,

GA4, GA7; [cytokinins] iP; [auxins] IAA; [stress signaling] ABA; [defense signaling]

SA, OPDA, JA-Ile) and volatile organic compound release (b-ionone, benzaldehyde, and

MeSA). Taken together, these increases in SA-defenses and phenolic/glucosinolate secondary metabolites suggest that insect feeding vibrations cause an upregulation of the biosynthesis pathway downstream of shikimic acid (phenylpropanoid and glucosinolate biosynthesis pathways; Figure 1.8).

Table 1.1 MSL proteins and their location in Arabidopsis thaliana plants. Proteins marked with an asterisk were studied in this work.

A. thaliana Protein Protein location Evidence (source) gene ID AT4G00290 MSL1 Mitochondria Experimental evidence (GFP tag; membrane Lee et al. 2016)

AT5G10490 MSL2 Chloroplast Experimental evidence (GFP tag; membrane Haswell and Meyerowitz 2006)

AT1G58200 MSL3 Chloroplast Experimental evidence (GFP tag; membrane Haswell and Meyerowitz 2006)

AT1G53470 MSL4 * Plasma membrane Predicted location (Haswell 2007)

AT3G14810 MSL5 * Plasma membrane Predicted location (Haswell 2007)

AT1G78610 MSL6 * Plasma membrane Predicted location (Haswell 2007)

AT2G17000 MSL7 Unknown Not reported

AT2G17010 MSL8 Pollen plasma Experimental evidence (GFP tag; membrane Hamilton et al. 2015)

AT5G19520 MSL9 * Plasma membrane, Experimental evidence (GFP tag; endoplasmic Haswell et al. 2008) reticulum

AT5G12080 MSL10 * Plasma membrane, Experimental evidence (GFP tag; endoplasmic Haswell et al. 2008) reticulum

Table 1.2. The effect of vibration and MeJA spray on secondary metabolite levels, based on a general linear mixed model. 'Round' (the set of plants tested at the same time) was included as a random effect. All p-values have been adjusted for testing of multiple metabolites, using the FDR procedure of Benjamini and Hochberg (1995). Significant effects are shown as follow: *** when p ≤ 0.001; ** when p ≤ 0.01; * when p ≤ 0.05; • when 0.05 ≤ p ≤ 0.10. N = 20 per treatment per genotype.

Secondary Metabolites Treatment D.F. F Value Pr > F Anthocyanins Vibe 1 22.53 0.000 *** MeJA 1 41.89 0.000 *** Genotype 2 3.02 0.051 • Vibe * MeJA 1 0.02 0.881 Genotype * Vibe 2 4.39 0.014 * Genotype * MeJA 2 0.08 0.926 Genotype * Vibe * MeJA 2 1.76 0.174 Flavonoids Vibe 1 27.69 0.000 *** MeJA 1 26.97 0.000 *** Genotype 2 4.90 0.008 ** Vibe * MeJA 1 0.75 0.387 Genotype * Vibe 2 6.94 0.001 *** Genotype * MeJA 2 0.42 0.659 Genotype * Vibe * MeJA 2 1.55 0.215 Total Phenolics Vibe 1 30.28 0.000 *** MeJA 1 7.50 0.007 ** Genotype 2 6.16 0.003 ** Vibe * MeJA 1 0.51 0.475 Genotype * Vibe 2 6.30 0.002 ** Genotype * MeJA 2 0.83 0.438 Genotype * Vibe * MeJA 2 1.10 0.336

Table 1.3. Table containing only statistical analysis for phytohormones that were involved in JA defense, SA defense, and ABA signaling. The effect of vibration and

MeJA spray on phytohormone levels, based on a general linear mixed model. 'Round'

(the set of plants tested at the same time) was included as a random effect. All p-values have been adjusted for testing of multiple hormones, using the FDR procedure of

Benjamini and Hochberg (1995). Significant effects are shown as follow: *** when p ≤

0.001; ** when p ≤ 0.01; * when p ≤ 0.05; • when 0.05 ≤ p ≤ 0.10. N = 15 per treatment per genotype. Abbreviations: ABA = 2-cis,4-trans-abscisic acid; SA = salicylic acid;

MeSA = methyl salicylate; OPDA = 13-epi-12-oxo-phytodienoic acid; JA = jasmonic acid; JA-Ile = jasmonoyl-isoleucine; MeJA = methyl jasmonate.

Phytohormones Treatment D.F. F Value Pr > F JA Defense Signaling OPDA Vibe 1, 168 2.24 0.136 MeJA 1, 168 0.41 0.521 Genotype 2, 168 1.76 0.176 Vibe * MeJA 1, 168 0.07 0.789 Genotype * Vibe 2, 168 0.74 0.477 Genotype * MeJA 2, 168 0.14 0.866 Genotype * Vibe * MeJA 2, 168 0.07 0.931 JA Vibe 1, 168 0.00 0.979 MeJA 1, 168 183.18 0.000 *** Genotype 2, 168 5.49 0.005 ** Vibe * MeJA 1, 168 3.23 0.074 • Genotype * Vibe 2, 168 2.52 0.084 • Genotype * MeJA 2, 168 2.63 0.075 • Genotype * Vibe * MeJA 2, 168 0.53 0.591 JA-Ile Vibe 1, 168 0.94 0.335 MeJA 1, 168 34.93 0.000 *** Genotype 2, 168 0.35 0.706 Vibe * MeJA 1, 168 0.14 0.705 Genotype * Vibe 2, 168 1.11 0.332 Genotype * MeJA 2, 168 0.67 0.511 Genotype * Vibe * MeJA 2, 168 0.59 0.556 37

MeJA Vibe 1, 168 0.17 0.683 MeJA 1, 168 0.67 0.415 Genotype 2, 168 2.38 0.097 • Vibe * MeJA 1, 168 0.02 0.885 Genotype * Vibe 2, 168 1.52 0.223 Genotype * MeJA 2, 168 2.12 0.125 Genotype * Vibe * MeJA 2, 168 0.28 0.758 SA Defense Signaling SA Vibe 1, 168 3.59 0.060 • MeJA 1, 168 2.99 0.085 • Genotype 2, 168 20.21 0.000 *** Vibe * MeJA 1, 168 1.00 0.320 Genotype * Vibe 2, 168 5.99 0.003 ** Genotype * MeJA 2, 168 1.48 0.231 Genotype * Vibe * MeJA 2, 168 0.01 0.995 MeSA Vibe 1, 168 1.85 0.176 MeJA 1, 168 0.42 0.518 Genotype 2, 168 1.92 0.150 Vibe * MeJA 1, 168 0.37 0.542 Genotype * Vibe 2, 168 2.55 0.081 • Genotype * MeJA 2, 168 1.55 0.215 Genotype * Vibe * MeJA 2, 168 .041 0.663 ABA Signaling ABA Vibe 1, 124 14.04 0.000 *** MeJA 1, 124 16.40 0.000 *** Genotype 2, 124 0.26 0.770 Vibe * MeJA 1, 124 4.54 0.035 * Genotype * Vibe 2, 124 0.76 0.471 Genotype * MeJA 2, 124 1.46 0.236 Genotype * Vibe * MeJA 2, 124 0.18 0.838

Figure 1.1. (A) Recording P. rapae feeding vibrations on A. thaliana using a laser vibrometer. (B) Feeding vibrations of P. rapae on A. thaliana, (top) waveform, (bottom) spectrogram. (C) Fourth-instar P. rapae caterpillar feeding on an A. thaliana leaf with a reflecting tape for the laser vibrometer to record the chewing vibrations produced. (D)

Healthy 5 week-old pre-reproductive A.s thaliana Rosette leaves are numbered in ascending order corresponding to age (from young to old), designating the youngest leaf larger than 6 mm as the first leaf. Leaf 7 (red number) was the target leaf for insect feeding vibration playback and chemical analyses. (E) Graphite rod mounted on a speaker is in contact with the underside of the leaf 7 (target leaf) of each plant to play back to the plant P. rapae vibrations previously recorded by Appel and Cocroft (2014).

(F) Each treatment and genotype is isolated from the others in a different box to prevent plant-to-plant communication via volatile organic compound release. (G) At 48 hours of incubation after treatment, the underside of the plants is turning purple due to anthocyanin accumulation. Target leaf 7 is marked with a red number. (H) Phytohormone

(bottom layer) extractions, with plant tissue located between both layers. (I) Phenolic (top layer) and pigment (bottom layer) extractions, with plant tissue located between the layers.

Figure 1.2. Comparisons showing direct, priming, and MeJA effects.

Figure 1.3. Phenolic concentrations (anthocyanins; flavonoids; total phenolics) in

Arabidopsis thaliana target leaf from each of the four treatments for the three genotypes

(wildtype, triple mutant msl4,5,6 and double mutant msl9,10). Data are represented in

µg/mg DW, as least square mean ± standard error. N = 20 per treatment per genotype.

Different letters (a, b, c, d) show statistically significant differences (p ≤ 0.05).

Figure 1.4. Photographs of the underside of A. thaliana (wildtype Col-0; double mutant msl9,10; triple mutant msl4,5,6) after two hours of sham or insect feeding vibrations followed by either water or MeJA spray. Pictures were taken after 48 hours of incubation.

Each target leaf (leaf 7 or 8) is marked by a red asterisk. The purple coloration of the underside of the leaves shows anthocyanin accumulation.

Figure 1.5. Concentrations of phytohormones involved in JA defense signaling in A. thaliana target leaf from each of the four treatments for the three genotypes (wildtype, triple mutant msl4,5,6 and double mutant msl9,10). Data are represented in ng/mg FW, as least square mean ± standard error. N = 15 per treatment per genotype. Different letters

(a, b, c) show statistically significant differences (p ≤ 0.05). Abbreviations: OPDA = 13- epi-12-oxo-phytodienoic acid; JA = jasmonic acid; JA-Ile = jasmonoyl-isoleucine; MeJA

= methyl jasmonate.

Figure 1.6. Concentrations of phytohormones involved in SA defense signaling in A. thaliana target leaf from each of the four treatments for the three genotypes (wildtype, triple mutant msl4,5,6 and double mutant msl9,10). Data are represented in ng/mg FW, as least square mean ± standard error. N = 15 per treatment per genotype. Different letters

(a, b, c) show statistically significant differences (p ≤ 0.05). Abbreviations: SA = salicylic acid, MeSA = methyl salicylate.

Figure 1.7. Concentration of phytohormone involved in ABA signaling of A. thaliana target leaf from each of the four treatments for the three genotypes (wildtype, triple mutant msl4,5,6 and double mutant msl9,10). Data are represented in ng/mg FW, as least square mean ± standard error. N = 15 per treatment per genotype. Different letters (a, b) show statistically significant differences (p ≤ 0.05). Abbreviations: ABA = abscisic acid.

Figure 1.8. Biosynthetic pathways of phytohormones and secondary metabolites. Arrows represent one or more steps in the biosynthesis. Compounds in bold have been analyzed in this study, and compounds in bold red displayed statistically significant vibration effect in one or more treatments/genotypes (see Results section for details). Figure adapted from Bruinsma et al. 2010; El Hadi et al. 2013; Niinemets et al. 2013.

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Supplements

Supplemental Table S1.1. UPLC-ESI-MS/MS parameters. Reaction monitoring conditions for protonated or deprotonated plant hormones ([M − H]+ or [M − H]–).

Abbreviations: GA1 = gibberellin 1; GA3 = gibberellic acid; GA4 = gibberellin 4; GA7 = gibberellin 7; iPR = 6-(Δ2-isopentenyl) adenine riboside; iP = 6-(Δ2-isopentenyl) adenine; tZR = trans-zeatin riboside; tZ = trans-zeatin; IAA = indole-3-acetic acid; IBA

= indole-3-butyric acid; ABA = 2-cis,4-trans-abscisic acid; SA = salicylic acid; MeSA = methyl salicylate; OPDA = 13-epi-12-oxo-phytodienoic acid; JA = jasmonic acid; JA-Ile

= jasmonoyl-isoleucine; MeJA = methyl jasmonate; D6-ABA, [2H6]- cis,trans-abscisic acid; D6-iP, [2H6]-N6-isopentenyladenine.

Phytohormones Scan Molecular Precursor ion Product ion Cone Collision Retention mode weight (g.mol-1) (Da) (Da) voltage (V) energy (V) time* (min) SA ES- 138.12 137.09 93.20 36 27 2.00 GA3 ES- 346.38 345.28 143.00 40 31 2.18 GA1 ES- 348.40 347.30 259.19 58 17 2.23 IAA ES- 175.19 174.09 130.02 32 15 2.38 D6-ABA ⦁ ES- 270.36 269.26 159.09 20 15 2.56 ABA ES- 264.32 263.22 152.99 26 13 2.56 IBA ES- 203.24 202.21 133.88 46 15 2.59 JA ES- 210.28 209.18 58.97 30 13 2.72 GA7 ES- 330.38 329.28 223.15 36 19 2.85 JA-Ile ES- 323.44 322.34 130.06 56 21 2.88 GA4 ES- 332.40 331.30 257.09 60 21 2.89 OPDA ES- 292.42 291.32 165.08 40 21 3.18 tZ ES+ 219.25 220.22 136.08 40 17 1.66 tZR ES+ 351.37 352.27 220.14 40 19 1.86 D6-iP ⦁ ES+ 209.29 210.26 137.05 36 13 2.18 iP ES+ 203.25 204.22 136.08 40 14 2.19 iPR ES+ 335.37 336.27 204.14 40 17 2.37 MeSA ES+ 152.15 153.12 121.00 36 11 3.33 MeJA ES+ 224.30 225.14 151.10 28 11 3.38

⦁ Internal standards.

* Retention times listed in this table are obtained under UPLC and column conditions mentioned in the protocol.

Supplemental Table S1.2. Water content of A. thaliana target leaf from each of the four treatments for the three genotypes (wildtype (WT), triple mutant msl4,5,6 and double mutant msl9,10). Data are represented in percentages, as average ± standard deviation. N

= 20 per treatment per genotype. No significant effect of treatments or genotypes was observed.

Sham*H2O Vibe*H2O Sham*MeJA Vibe*MeJA Water content WT 87.85 ± 0.70 87.29 ± 0.91 87.14 ± 1.26 87.51 ± 1.39 msl4,5,6 87.87 ± 0.72 87.41 ± 0.93 86.98 ± 1.10 87.11 ± 1.06 msl9,10 87.74 ± 1.64 86.71 ± 2.63 88.36 ± 1.23 86.66 ± 1.42

Supplemental Table S1.3. Secondary metabolite concentrations (anthocyanins; flavonoids; total phenolics) of A. thaliana target leaf from each of the four treatments for the three genotypes (wildtype (WT), triple mutant msl4,5,6 and double mutant msl9,10).

Data are represented in µg/mg DW, as least square mean ± standard error. N = 20 per treatment per genotype.

Secondary Metabolites Sham*H2O Vibe*H2O Sham*MeJA Vibe*MeJA Anthocyanins WT 0749.78 ± 66.35 0940.02 ± 64.81 0896.93 ± 65.09 1143.49 ± 66.18 msl4,5,6 0884.60 ± 69.60 0973.46 ± 67.21 1157.76 ± 70.74 1108.45 ± 67.35 msl9,10 0809.67 ± 67.89 0897.80 ± 66.18 0936.45 ± 70.49 1131.74 ± 70.53 Flavonoids WT 141.64 ± 6.16 161.25 ± 6.02 151.88 ± 6.05 183.43 ± 6.15 msl4,5,6 159.26 ± 6.48 164.63 ± 6.24 179.83 ± 6.59 176.44 ± 6.26 msl9,10 156.51 ± 6.30 165.30 ± 6.15 162.07 ± 6.56 181.89 ± 6.56 Total Phenolics WT 095.19 ± 4.00 107.2 ± 3.90 096.72 ± 3.92 117.39 ± 3.99 msl4,5,6 105.53 ± 4.20 108.97 ± 4.05 114.43 ± 4.27 113.86 ± 4.06 msl9,10 105.42 ± 4.09 113.37 ± 3.99 105.84 ± 4.25 116.58 ± 4.25

Supplemental Table S1.4. Pigment content (total carotenoids; chlorophylls a and b) of

A. thaliana target leaf from each of the four treatments for the three genotypes (wildtype

(WT), triple mutant msl4,5,6 and double mutant msl9,10). Data are represented in mg/mg

DW, as least square mean ± standard error. N = 20 per treatment per genotype. No significant effect of treatments or genotypes was observed.

Pigments Sham*H2O Vibe*H2O Sham*MeJA Vibe*MeJA Carotenoids WT 2.153 ± 0.179 2.128 ± 0.177 2.188 ± 0.177 2.265 ± 0.179 msl4,5,6 2.298 ± 0.183 2.049 ± 0.180 2.285 ± 0.184 2.340 ± 0.181 msl9,10 2.412 ± 0.181 2.133 ± 0.179 2.221 ± 0.184 2.137 ± 0.184 Chlorophylls WT 4.338 ± 0.138 3.991 ± 0.135 4.196 ± 0.136 4.098 ± 0.138 msl4,5,6 4.362 ± 0.145 4.183 ± 0.140 4.353 ± 0.147 4.277 ± 0.140 msl9,10 4.370 ± 0.141 3.748 ± 0.138 4.115 ± 0.147 4.042 ± 0.147

Supplemental Table S1.5. Concentrations of growth regulators (gibberellins; cytokinins; auxins), stress signaling (abscisic acid), SA and JA defense signaling of A. thaliana target leaf from each of the four treatments for the three genotypes (wildtype (WT), triple mutant msl4,5,6 and double mutant msl9,10). Data are represented in ng/mg FW, as least square mean ± standard error. N = 15 per treatment per genotype. Abbreviations: GA1 = gibberellin 1; GA3 = gibberellic acid; GA4 = gibberellin 4; GA7 = gibberellin 7; iPR = 6-

(Δ2-isopentenyl) adenine riboside; iP = 6-(Δ2-isopentenyl) adenine; tZR = trans-zeatin riboside; tZ = trans-zeatin; IAA = indole-3-acetic acid; IBA = indole-3-butyric acid;

ABA = 2-cis,4-trans-abscisic acid; SA = salicylic acid; MeSA = methyl salicylate; OPDA

= 13-epi-12-oxo-phytodienoic acid; JA = jasmonic acid; JA-Ile = jasmonoyl-isoleucine;

MeJA = methyl jasmonate.

Phytohormones Sham*H2O Vibe*H2O Sham*MeJA Vibe*MeJA Gibberellins GA1 WT 96.45 ± 5.18 97.13 ± 5.22 106.30 ± 5.71 108.90 ± 5.85 msl4,5,6 97.47 ± 5.24 93.47 ± 5.33 95.97 ± 5.76 95.44 ± 5.14 msl9,10 97.38 ± 5.31 95.10 ± 5.26 101.80 ± 5.62 99.75 ± 5.46 GA3 WT 1.251 ± 0.178 1.041 ± 0.148 1.285 ± 0.183 1.483 ± 0.211 msl4,5,6 1.225 ± 0.169 1.170 ± 0.172 1.156 ± 0.177 0.989 ± 0.132 msl9,10 1.286 ± 0.183 1.205 ± 0.172 1.202 ± 0.171 1.149 ± 0.164 GA4 WT 0.628 ± 0.138 0.630 ± 0.139 0.720 ± 0.159 1.837 ± 0.405 msl4,5,6 0.675 ± 0.144 0.456 ± 0.104 0.766 ± 0.181 0.563 ± 0.117 msl9,10 0.490 ± 0.108 0.859 ± 0.189 0.554 ± 0.122 0.453 ± 0.100 GA7 WT 0.047 ± 0.012 0.081 ± 0.020 0.093 ± 0.023 0.082 ± 0.020 msl4,5,6 0.117 ± 0.029 0.115 ± 0.030 0.106 ± 0.028 0.127 ± 0.030 msl9,10 0.102 ± 0.026 0.092 ± 0.023 0.058 ± 0.014 0.059 ± 0.015 Cytokinins iPR WT 7.999 ± 0.620 8.060 ± 0.625 7.948 ± 0.616 8.332 ± 0.646 msl4,5,6 7.789 ± 0.596 7.395 ± 0.603 7.191 ± 0.615 6.168 ± 0.469 msl9,10 7.831 ± 0.612 7.631 ± 0.602 7.172 ± 0.565 7.566 ± 0.592 iP WT 0.183 ± 0.020 0.172 ± 0.019 0.174 ± 0.019 0.205 ± 0.022 msl4,5,6 0.201 ± 0.021 0.158 ± 0.018 0.169 ± 0.020 0.149 ± 0.015 msl9,10 0.183 ± 0.020 0.167 ± 0.018 0.247 ± 0.027 0.170 ± 0.018

69 tZR WT 1.788 ± 0.211 1.775 ± 0.209 1.859 ± 0.219 1.880 ± 0.221 msl4,5,6 1.448 ± 0.172 1.650 ± 0.208 1.576 ± 0.208 1.246 ± 0.149 msl9,10 1.632 ± 0.196 1.618 ± 0.198 1.605 ± 0.196 1.216 ± 0.147 tZ WT 0.342 ± 0.050 0.329 ± 0.048 0.286 ± 0.042 0.378 ± 0.055 msl4,5,6 0.336 ± 0.048 0.292 ± 0.044 0.315 ± 0.049 0.300 ± 0.041 msl9,10 0.376 ± 0.055 0.366 ± 0.053 0.350 ± 0.051 0.304 ± 0.044 Auxins IAA WT 21.32 ± 4.52 28.98 ± 6.14 21.67 ± 4.59 28.96 ± 6.14 msl4,5,6 15.37 ± 3.26 15.13 ± 3.33 25.51 ± 5.61 15.16 ± 3.01 msl9,10 13.58 ± 2.88 18.10 ± 3.84 15.51 ± 3.41 18.72 ± 4.12 IBA WT 0.095 ± 0.011 0.100 ± 0.011 0.095 ± 0.011 0.132 ± 0.015 msl4,5,6 0.113 ± 0.013 0.098 ± 0.012 0.099 ± 0.013 0.096 ± 0.010 msl9,10 0.123 ± 0.014 0.100 ± 0.012 0.079 ± 0.009 0.097 ± 0.011 ABA Signaling ABA WT 0.0073 ± 0.0004 0.0077 ± 0.0004 0.0074 ± 0.0004 0.0094 ± 0.0005 msl4,5,6 0.0072 ± 0.0004 0.0078 ± 0.0005 0.0073 ± 0.0004 0.0087 ± 0.0005 msl9,10 0.0072 ± 0.0004 0.0073 ± 0.0004 0.0082 ± 0.0005 0.0092 ± 0.0005 SA Defense Signaling SA WT 0.053 ± 0.006 0.089 ± 0.011 0.074 ± 0.009 0.111 ± 0.014 msl4,5,6 0.051 ± 0.006 0.049 ± 0.006 0.061 ± 0.008 0.051 ± 0.006 msl9,10 0.044 ± 0.005 0.050 ± 0.006 0.047 ± 0.006 0.045 ± 0.006

MeSA WT 1.629 ± 0.317 1.543 ± 0.300 1.222 ± 0.238 1.202 ± 0.234 msl4,5,6 1.834 ± 0.267 1.367 ± 0.314 2.761 ± 0.259 1.329 ± 0.243 msl9,10 1.419 ± 0.356 1.561 ± 0.266 1.240 ± 0.537 1.348 ± 0.262 JA Defense Signaling OPDA WT 0.707 ± 0.108 0.779 ± 0.119 0.684 ± 0.105 0.821 ± 0.126 msl4,5,6 0.833 ± 0.124 1.103 ± 0.176 0.771 ± 0.127 0.978 ± 0.141 msl9,10 0.889 ± 0.136 0.842 ± 0.129 0.780 ± 0.120 0.818 ± 0.125 JA WT 0.049 ± 0.006 0.054 ± 0.006 0.122 ± 0.013 0.088 ± 0.010 msl4,5,6 0.043 ± 0.005 0.043 ± 0.005 0.117 ± 0.014 0.097 ± 0.010 msl9,10 0.054 ± 0.005 0.059 ± 0.006 0.138 ± 0.016 0.162 ± 0.019 JA-Ile WT 0.00044 ± 0.00006 0.00048 ± 0.00006 0.00071 ± 0.00010 0.00081 ± 0.00010 msl4,5,6 0.00041 ± 0.00005 0.00053 ± 0.00007 0.00061 ± 0.00009 0.00069 ± 0.00009 msl9,10 0.00050 ± 0.00007 0.00040 ± 0.00005 0.00074 ± 0.00010 0.00078 ± 0.00010 MeJA WT 0.359 ± 0.051 0.391 ± 0.056 0.329 ± 0.047 0.355 ± 0.051 msl4,5,6 0.318 ± 0.046 0.311 ± 0.047 0.305 ± 0.049 0.331 ± 0.048 msl9,10 0.389 ± 0.046 0.343 ± 0.051 0.549 ± 0.081 0.412 ± 0.060