Mechanosensation in Plants by Taylor Y. Paret Submitted to the Graduate
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A Thesis entitled To Hear Without an Ear: Mechanosensation in Plants by 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 Copyright 2019, Taylor York Paret 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. by 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. iv 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. 1 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 2 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 3 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. 4 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. 5 List of Supplements Supplemental Table