Iowa State University Capstones, Theses and Graduate Theses and Dissertations Dissertations 2019 Integrated ‘omics’ characterization of conserved nectar production mechanisms using floral and extrafloral eudicot nectaries Elizabeth Claire Chatt Iowa State University Follow this and additional works at: https://lib.dr.iastate.edu/etd Part of the Agriculture Commons, and the Plant Sciences Commons Recommended Citation Chatt, Elizabeth Claire, "Integrated ‘omics’ characterization of conserved nectar production mechanisms using floral and extrafloral eudicot nectaries" (2019). Graduate Theses and Dissertations. 17154. https://lib.dr.iastate.edu/etd/17154 This Dissertation is brought to you for free and open access by the Iowa State University Capstones, Theses and Dissertations at Iowa State University Digital Repository. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Integrated ‘omics’ characterization of conserved nectar production mechanisms using floral and extrafloral eudicot nectaries by Elizabeth Claire Chatt A dissertation submitted to the graduate faculty in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Major: Plant Biology Program of Study Committee: Basil J. Nikolau, Major Professor Harry T. Horner Daniel S. Nettleton Jonathan F. Wendel Eve Syrkin Wurtele Marna D. Yandeau-Nelson The student author, whose presentation of the scholarship herein was approved by the program of study committee, is solely responsible for the content of this dissertation. The Graduate College will ensure this dissertation is globally accessible and will not permit alterations after a degree is conferred. Iowa State University Ames, Iowa 2019 Copyright © Elizabeth Claire Chatt, 2019. All rights reserved. ii TABLE OF CONTENTS ABSTRACT…………………………………………………………………………. iii CHAPTER 1. GENERAL INTRODUCTION………………………………………. 1 References……………………………………………………………………. 4 CHAPTER 2. SEX-DEPENDENT VARIATION OF PUMPKIN (CUCURBITA MAXIMA CV. BIG MAX) NECTAR AND NECTARIES AS DETERMINED BY PROTEOMICS AND METABOLOMICS………………………………………….. 9 Abstract………………………………………………………………………. 9 Introduction…………………………………………………………………... 10 Materials and Methods……………………………………………………….. 12 Results………………………………………………………………………... 19 Discussion……………………………………………………………………. 25 Conclusion…………………………………………………………………… 34 Author Contributions………………………………………………………… 34 Funding………………………………………………………………………. 34 Acknowledgements…………………………………………………………... 34 References……………………………………………………………………. 35 CHAPTER 3. COMPARATIVE TRANSCRIPTOMICS, METABOLOMICS, AND ULTRASTRUCTURAL ANALYSES CHARACTERIZE KEY MODULES OF NECTAR SYNTHESIS AND SECRETION OF COTTON (GOSSYPIUM HIRSUTUM) FLORAL AND EXTRAFLORAL NECTARIES……………………. 40 Introduction…………………………………………………………………... 40 Materials and Methods……………………………………………………….. 43 Results………………………………………………………………………... 52 Discussion……………………………………………………………………. 65 Author Contributions………………………………………………………… 75 Acknowledgements and Funding…………………………………………….. 76 References……………………………………………………………………. 76 Tables and Figures…………………………………………………………… 84 CHAPTER 4. METABOLOMIC PROFILING OF NICOTIANA SPP. NECTARS REVEALS THE INTERPLAY BETWEEN PLANT PHYLOGENETICS AND POLLINATORS IN SHAPING NECTAR DIVERSIFICATION…………………... 104 Abstract………………………………………………………………………. 104 Introduction…………………………………………………………………... 105 Materials and Methods……………………………………………………….. 107 Results………………………………………………………………………... 110 Discussion……………………………………………………………………. 115 Author Contributions………………………………………………………… 119 Acknowledgements…………………………………………………………... 120 Data Accessibility……………………………………………………………. 120 References……………………………………………………………………. 120 Tables and Figures…………………………………………………………… 126 CHAPTER 5. GENERAL CONCLUSION…………………………………………. 134 References……………………………………………………………………. 138 iii ABSTRACT Floral and extrafloral nectar, produced by nectaries, is offered as a reward to foster plant-animal mutualisms with pollinators and invertebrate predators. Attraction of pollinators through floral nectar improves fruit set in 87 out of 115 global food crops. Meanwhile extrafloral nectar, reported in 745 genera, attracts invertebrate predators, such as ants, as an indirect defense mechanism to reduce herbivory. Nectar quality (i.e. volume and composition) strongly correlates with the efficiency of these plant-animal mutualistic interactions, yet nectar composition has typically only been defined by targeted analyses of the two most predominant classes of metabolites, carbohydrates and amino acids. Other less abundant components of nectar are often unaccounted (i.e. vitamins, alkaloids, phenolics, terpenoids, lipids, metal ions, hormones, and proteins). Furthermore, molecular understanding of nectar synthesis and secretion is limited to a few reports of genes directly affecting the de novo production or quality of floral nectar. Comprehensive GC-MS based metabolomics techniques capable of quantifying trace components of nectar were used to characterize nectar composition from species, spanning three eudicot families (Cucurbitaceae, Malvaceae, and Solanaceae). This enabled examination of relationships between nectar composition and biological factors such as the sex of the flower, plant-animal mutualisms, and functional role of the nectar regarding plant reproductive success and defense (i.e. floral and extrafloral nectar). These analyses contributed the metabolomics portion of a comprehensive systems network-based project to define the conserved molecular mechanisms of nectar synthesis and secretion among floral and extrafloral nectaries of the core eudicots. Through the analysis of the transcriptomes and proteomes of nectaries from a broad range of phylogenetic plant clades, we identified core sets of genes conserved within eudicots required for nectary synthesis and secretion. These results also supply a foundation for targeted studies of nectar quality improvement, which will benefit pollinator health, promote plant reproductive success, and enhances biological control of crop pests. 1 CHAPTER 1: GENERAL INTRODUCTION Nectaries are specialized glandular tissues of plants first recognized by Linnaeus (1758) that function to produce and secrete nectars, sugar-rich solutions. Nectaries present on a recognized floral structure are referred to as floral nectaries, whereas nectaries developing outside of the flower (stems, petioles, leaves etc.) are referred to as extrafloral nectaries. The secreted nectars are presented as rewards to animal mutualists in exchange for the ecosystem service of pollination in the case of floral nectar and indirect resistance to herbivores by recruiting pugnacious predatory insects to the extrafloral nectar (Chamberlain and Rudgers, 2012; Mitchell et al., 2009; Ollerton, 2017; Wäckers et al., 2001). The patterns of nectar secretion vary between floral and extrafloral nectaries in order to optimize benefits while minimizing the energetic cost of producing nectar (Heil, 2011; Pleasants, 1983; Wäckers and Bonifay, 2004). The floral nectaries typically produce nectar during anthesis, whereas the extrafloral nectaries modulate nectar secretion based on environmental stressors such as insect herbivory (Heil, 2015; Wäckers et al., 2001). Nearly 75% of our global food crops depend on or benefit from animal-mediated pollination commonly facilitated by floral nectar which improves seed set and promotes outcrossing (Klein et al., 2007). Even in cotton, a largely self- pollinated crop, honey bee visitation facilitated by the floral nectar increases yield (Rhodes, 2002). In a variety of crops such as oilseed rape, sunflower, pumpkin, and tobacco, variations in nectar composition, viscosity, and volume directly influence the frequency of pollinator visitation (Carruthers et al., 2017; Mallinger and Prasifka, 2017; Nepi and Pacini, 1993; Raguso et al., 2003). More generally, nectar composition often reflects the feeding preferences 2 of the target animal-mutualist shaped by factors such as dietary requirements to sustain foraging and neuronal response to phagostimulatory metabolites. (Baker and Baker, 1983; Chen and Welch, 2014; Gardener and Gillman, 2002; Hendriksma et al., 2014; Waller, 1972). The most conserved classes of nectar metabolites are carbohydrates, predominately fructose, glucose, and sucrose, and amino acids which are present at concentrations a thousand-fold less than the carbohydrates (Lüttge, 1977; Nicolson and Thornburg, 2007). Thus, nectar ecology studies typically define nectar composition based upon targeted analyses of predominant sugars and occasionally the amino acids. The ratios of the predominate nectar sugars are the primary means of classifying nectars, into four categories: hexose-dominant, hexose-rich, sucrose-rich, and sucrose-dominant (Baker and Baker, 1983). In addition to carbohydrates and amino acids, nectars are complex solutions that contain some or all of the following constituents: vitamins, alkaloids, phenolics, terpenoids, lipids, metal ions, hormones, and proteins (Richardson et al., 2015; Roy et al., 2017). Global assessments of these ‘trace’ nectar metabolites using metabolomics techniques is a recent development (Bender et al., 2012, 2013; Kram et al., 2008; Noutsos et al., 2015). The hypothesized mechanisms of nectar production and secretion are historically based on ultrastructural analyses, which have generated two models: (1) merocrine also referred to as granulocrine and (2) eccrine (Fahn,