City University of New York (CUNY) CUNY Academic Works Dissertations, Theses, and Capstone Projects CUNY Graduate Center 9-2021 Three LC-MS Plant Metabolomics Studies of Hop (Humulus) Species: Wild H. neomexicanus, Drought Stress, and Agricultural Terroir Taylan Morcol The Graduate Center, City University of New York How does access to this work benefit ou?y Let us know! More information about this work at: https://academicworks.cuny.edu/gc_etds/4443 Discover additional works at: https://academicworks.cuny.edu This work is made publicly available by the City University of New York (CUNY). Contact: [email protected] THREE LC-MS PLANT METABOLOMICS STUDIES OF HOP (HUMULUS) SPECIES: WILD H. NEOMEXICANUS, DROUGHT STRESS, AND AGRICULTURAL TERROIR by TAYLAN BARIŞ MORÇÖL A dissertation submitted to the Graduate Faculty in Biology in partial fulfillment of the requirements for the degree of Doctor of Philosophy, The City University of New York 2021 © 2021 TAYLAN BARIŞ MORÇÖL All Rights Reserved ii Three LC-MS Plant Metabolomics Studies of Hop (Humulus) Species: Wild H. neomexicanus, Drought Stress, and Agricultural Terroir by Taylan Barış Morçöl This manuscript has been read and accepted for the Graduate Faculty in Biology in satisfaction of the dissertation requirement for the degree of Doctor of Philosophy. ____________________ _______________________________________ Date Renuka P. Sankaran Chair of Examining Committee ____________________ _______________________________________ Date Cathy Savage-Dunn Executive Officer Supervisory Committee: Edward J. Kennelly, Advisor Paul D. Matthews Renuka P. Sankaran Damon Little Michael Balick THE CITY UNIVERSITY OF NEW YORK iii ABSTRACT Three LC-MS Plant Metabolomics Studies of Hop (Humulus) Species: Wild H. neomexicanus, Drought Stress, and Agricultural Terroir by Taylan Barış Morçöl Advisor: Edward J. Kennelly The hop plant (Humulus L., Cannabaceae) is a dioecious, perennial, twining vine with a long history of human use. Nowadays, hop plants are generally grown for their inflorescences (“cones”), which are used in brewing for their phytochemical metabolites. Many of these metabolites are involved in plant stress response and communication. Genetics and environment are two major factors that affect plant metabolism. In three separate metabolomics studies, this project examined the effects of both genetic and environmental factors on hop phytochemistry. In the first study, 23 hop genotypes were grown in two different locations in the Pacific Northwest region of the United States. Each location has a distinct set of environmental conditions or terroir. Ultrahigh- performance liquid chromatography–tandem mass spectrometry (UPLC-MS/MS) was used to measure the relative amounts of four glycosylated aroma compounds in hop cones previously processed to remove resins and oils. Analysis of variance (ANOVA) showed that the large majority of the variability in levels of linalool, raspberry ketone, and 2-phenylethanol glucosides was attributable to genotype differences. In contrast, difference in growing location had the largest effect on levels of the glycosylated green leaf iv volatile, hexyl glucoside, although the effect of genetics was still substantial. While the impact of hop aroma glycosides on beer aroma is considered small relative to that of hop essential oils, the influences of genotype and environment on hop aroma glycoside content may still have some effect on beer aroma development as it ages. In the second study, three levels of water stress were replicated across four hop genotypes in a controlled greenhouse experiment. Untargeted UPLC-QTof-MSE was used to profile a wide range of metabolites in the leaves. Extensive filtering reduced the number of chemical features from 2387 to 278. Principal component analysis (PCA) of the filtered features showed that the drought treatments had a noticeable effect on the overall chemical composition of most genotypes. Still, each genotype appeared distinct, suggesting that genotype (rather than drought stress) had the larger effect on hop leaf chemical composition. Also, the study found 14 phytochemical markers that consistently increased or decreased in response to water stress. Ten of these markers were tentatively identified: five monoacylated glycerolipids, two norisoprenoids (abscisic acid [ABA] and roseoside), pheophorbide A, glutaric acid, and dihydromyricetin. All but dihydromyricetin were more abundant in drought stressed leaves. The glycerolipids, norisoprenoids, and pheophorbide A all have known or likely stress signaling roles, and their accumulation likely indicates respective breakdowns of membrane lipids, carotenoids, and chlorophyll. Many of these compounds are likely drought stress markers in higher plants in general, while some may be more narrow or depend on the type of drought treatment. In the third study, UPLC-MS was used to compare differences in leaf phytochemical composition and glandular trichome density between two groups of greenhouse-grown hop plants representing two distinct genetic lineages: commercial hop cultivars of pure European or mixed European–North American heritage; and wild hops from the American Southwest (H. neomexicanus). In an untargeted principal component analysis, the phytochemical compositions of the two groups appeared distinct. Bitter acid and prenylflavonoid levels were higher in the H. neomexicanus group; so was the density of glandular trichomes, where bitter acids and prenylflavonoids are biosynthesized. The H. neomexicanus group also v had higher flavonol glycoside levels. However, all six phenolic acids measured were higher in the cultivar group, although only one of these differences was statistically significant. Due to the higher levels of bitter acids, prenylflavonoids, and flavonol glycosides in their leaves, the H. neomexicanus genotypes may have greater resistance to insect herbivory, fungal infection, or both and should be evaluated for these characteristics. vi ACKNOWLEDGMENTS In some of the early presentations that I gave during this PhD program, I had several slides titled “It takes a village…”. This comes from the famous African proverb, “It takes a village to raise a child.” Although this PhD degree is in my name, this achievement has only been possible though the efforts of a whole community of people in my life. With that in mind, I acknowledge several people and organizations. First of all, my committee members. Dr. Michael Balick was my inspiration for applying to the CUNY/NYBG PhD program in the first place. I was inspired by Dr. Balick’s decades of ethnobotanical work. Although ethnobotany was not the main focus of my doctoral research, I value the perspective that Dr. Balick has brought to my work, helping me see the cultural and economic context of my phytochemical research. In addition, Dr. Balick has been so willing to connect me with his broad professional network. Dr. Renuka Sankaran’s critical feedback has always been insightful and has helped strengthen my scientific work. I also appreciate Renuka’s friendship over the years. I am honored for the many times that she invited me into her home and shared food with me. It has been a lot of fun getting to know her family and especially watching her kids grow up. Dr. Damon Little joined my committee when I was looking for someone to advise on my population genetics work, which ultimately did not make it into the dissertation. Nonetheless, Damon’s insights about pop-gen and statistics, neither of which I had much experience with at the start, were so helpful. During my meetings with him, Damon would patiently explain new concepts to me and answer my questions. I always walked away from these meetings feeling refreshed with a new perspective. His friendly and encouraging demeanor helped me keep the challenges of grad school in perspective. Dr. Paul Matthews has been a huge part of my life these past several years. I am so grateful that Paul decided to take me under his wing and offer me a fully-funded graduate fellowship within his research vii program at Hopsteiner. Early on, Paul and I had a some conversations that helped us discover common ground in our research interests. These formative conversations led to the development of my southwestern American wild hop research plan within Paul’s vision of a global, wild hop conservation project. Over the years, Paul and I have road tripped, camped, and hiked together, all in search of wild hops. Paul has also hosted me several times in his own home and introduced me to his family. And this is all on top of Paul’s continual intellectual guidance, which has helped keep my academic research relevant to the hop industry. I am truly grateful for Paul’s mentorship. The efforts of many other people at Hopsteiner also played a part in my success. I am especially grateful to Harald Schwarz and Troy Elliot for their continual financial support despite slow progress in the early days of my project. I also thank several members of Dr. Paul Matthews’ research and development team for their intellectual and logistical support as well as their friendship and hospitality: Nicholi Pitra, Mark Coles, Tiffany Pitra, Rachel Bussey, Dr. Dong Zhang, Dr. Alexander Feiner, Dr. Nicholas Price, Joshua Havill, Mohammad Mafakheri, Nicholas Hansen, James Robertson, Samantha Ferreira, Dr. Martin Biendl, Dr. Christina Schmidt, Dr. Martin Steinhauser, and Dr. Katherine Easterling. Katherine Easterling’s assistance, friendship, and famous tofu dip recipe during one of my wild hop collecting trips was