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Ecology and Economic Potential of Secondary Metabolites Produced by Yaupon Holly, Ilex vomitoria

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Title:
Ecology and Economic Potential of Secondary Metabolites Produced by Yaupon Holly, Ilex vomitoria
Creator:
PALUMBO, MATTHEW J. ( Author, Primary )
Copyright Date:
2008

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Subjects / Keywords:
Alkaloids ( jstor )
Antioxidants ( jstor )
Beverages ( jstor )
Fertilization ( jstor )
Leaves ( jstor )
Metabolism ( jstor )
Metabolites ( jstor )
Nitrogen ( jstor )
Plants ( jstor )
Tea ( jstor )

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University of Florida
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University of Florida
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Copyright Matthew J. Palumbo. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Embargo Date:
6/30/2007
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659561081 ( OCLC )

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1 ECOLOGY AND ECONOMIC POTENTIAL OF SECONDARY METABOLITES PRODUCED BY YAUPON HOLLY, Ilex vomitoria By MATTHEW J. PALUMBO A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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2 Copyright 2006 by Matthew J. Palumbo

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3 To my mother who has s upported my every interest Â…and to the memory of my father who instilled in me a deep respect for Nature.

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4 ACKNOWLEDGMENTS I would like to thank my advi sor Jack Putz who not only handed me a great master’s project and helped me get into graduate school but who also kept on my ass and stuck with me throughout this process despite my persistent bitching and moaning and tortoise-like work pace. Jack and his family, Claudia, Juliana, and Ant onio have graciously welcomed me (and my dog!) into their home and on their beautiful land on ma ny occasions, which made this an especially enjoyable graduate school experience. I would also like to thank Steve Talcott, who although is not now an official member of my committee, has nevertheless been a great chemistry teacher and an invaluable source of knowledge that has greatly enhanced my Master’s project. I would also like to thank Michelle Mack who sat on my master's committee for providing useful comments on my research and manuscript drafts a nd Rick Stepp for his willingness to join my committee at such a late stage of my master’s project. I also thank other faculty members in th e Department of Botany who enhanced my education. In particular, I thank Walter Judd whose taxonomy classes were amazing whirlwinds through the incredible floral diversity on th is planet; and Steve Mulkey whose plant ecophysiology course gave me a broader perspec tive of the physiological and evolutionary mechanisms inherent to the study of ecology. My fellow graduate student s were also a great help and inspiration. I particularly thank Mo rgan Varner for helping me to understand the dynamics of the first ecosystem that I ever real ly came to (somewhat) understand. I also thank Eddie Watkins for teaching me how to measure light with a fish-eye lens and computer software, which has become a critical component of my research. I thank Kim and Jorge (from Steve Talcott’s lab) for helping me get thro ugh my last round of caffeine assays. André Khuri from the Department of Statisti cs provided invaluable assistance interpreting data for chapter 1. I also thank Jenny Schafer for “teaching” me how to use Sigma Plot, which

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5 was used to plot the graphs in chapter 1. A nd I thank Paulo Brando, the new ‘R’ guru in Jack Putz’s lab, for last-minute help with statistics and graphing. I also tha nk Ann Wagner who is the botany lab manager for her help and patience with my technological ineptitude. Also thanks to Isabel Meister for help with se tting up the campus-plant study. I also thank all my friends in Gainesville from the Department of Botany, other departments at the University of Florida, and elsewhere. Without the sharing of experience that comes with fr iendship, this degree would have been impossible. I particularly want to thank Jason and Nicole Frederick for all their support and for providing me with a home (and not just a place to lay my head) here in Gainesville. I would like to thank those students in my la boratory sections and le ctures who made my TA-ship a rewarding way to pay the bills. My gratitude goes to these students for their forgiveness, willingness to put up with my idiosy ncrasies, laughter at my pathetic attempt at jokes, and the general ch allenge they represented. I only hope th ey learned as much from me as I did from them. Finally, I thank my family. I thank my brother who encouraged me to return to school when I was only considering it 6 years ago. I thank my grandfather w ho never hesitated with financial assistance for my education when it wa s needed and who has allowed me to share my passion for plants and ecology with him. And I thank my mother who always supported me and encouraged me to stay with the program on da ys when it felt overwhelming. I can never thank her enough. Finally, I thank Vito, this man’s best friend, w ho always greets me at the end of the day with his smiling enthusiasm and zest for life whether I can return it or not.

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6 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........7 LIST OF FIGURES................................................................................................................ .........8 CHAPTER 1 NITROGEN FERTILIZER AND GE NDER EFFECTS ON THE SECONDARY METABOLISM OF YAUPON, A CAFFEIN E-CONTAINING NORTH AMERICAN HOLLY.......................................................................................................................... .........11 Introduction................................................................................................................... ..........11 Materials and Methods.......................................................................................................... .14 Plant Material and Nitrogen Fertilization........................................................................14 Chemical Analyses..........................................................................................................15 Statistical Analyses..........................................................................................................1 6 Results........................................................................................................................ .............16 Methylxanthine Alkaloids...............................................................................................16 Phenolics...................................................................................................................... ....16 Nitrogen Content.............................................................................................................17 Antioxidant Capacity.......................................................................................................18 Discussion..................................................................................................................... ..........18 Conclusions.................................................................................................................... .........24 2 Ilex vomitoria : AN OVERLOOKED NORTH AM ERICAN CAFFEINE SOURCE............28 Introduction................................................................................................................... ..........28 Caffeine and Conservation.....................................................................................................2 8 History of Yaupon Tea....................................................................................................28 Yaupon Chemistry and Production.................................................................................31 Materials and Methods.......................................................................................................... .33 Experimental Design.......................................................................................................33 Chemical Analyses..........................................................................................................33 Measurement of Light Availability.................................................................................35 Statistical Analysis..........................................................................................................3 5 Results........................................................................................................................ .............35 Conclusions.................................................................................................................... .........38 LIST OF REFERENCES............................................................................................................. ..47 BIOGRAPHICAL SKETCH.........................................................................................................54

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7 LIST OF TABLES Table page 1-1 ANOVA results for foliar concentrations of methylxanthine (MX) alkaloids in Ilex vomitoria ............................................................................................................................25 1-2 ANOVA results for foliar concentrations of phenolic compounds in Ilex vomitoria ........25 2-1 Mean ± 1 SE ORAC (oxygen radical ab sorbance capacity) values for yaupon holly ( Ilex vomitoria, variety ‘Nana’ ) , wild –type yaupon ( I. vomitoria ), yerba maté ( I. paraguariensis), and green tea ( Camellia sinensis )...........................................................46

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8 LIST OF FIGURES Figure page 1-1 Mean (+1 SE) foliar concentrations of alkaloids and nitrogen by treatment and gender in Ilex vomitoria (note difference in scales)...........................................................26 1-2 Mean (+1 SE) foliar concentrations of phenolics and antioxidant capacity by treatment and gender in Ilex vomitoria (note difference in scales)....................................27 2-1 Cultivated hedge of yaupon ( Ilex vomitoria ) variety ‘Nana’ on the University of Florida campus, Gaines ville, Florida.................................................................................40 2-2 Mean (+/SE) A) leaf mass, B) caffeine concentration, and C) total caffeine yield in control and fertilized Ilex vomitoria cultivar ‘Nana’ individuals obtained from the second harvest of this study...............................................................................................41 2-3 Mean (+/SE) A)antioxidant capacity and B) total antioxidant capacity in control and fertilized Ilex vomitoria cultivar ‘Nana’ individuals obtained from the second harvest of this study ......................................................................................................... .42 2-4 Relationship between % full sun and ca ffeine concentration (% dry weight of foliage) for yaupon holly ( Ilex vomitoria ) cultivar ‘Nana.’ Line indicates linear regression. ................................................................................................................... ......43 2-5 Relationship between % full sun and antio xidant capacity, as measured by the ORAC method, for yaupon holly ( Ilex vomitoria ) cultivar ‘Nana.’ Line indicates linear regression. .................................................................................................................. ......44 2-6 Mean (+1 SE) foliar caffeine con centrations in f our varieties of Ilex vomitoria . Lower case letters specify groupings as indicated by Tukey’s Tests for multiple comparisons.................................................................................................................... ...45

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9 Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science ECOLOGY AND ECONOMIC POTENTIAL OF SECONDARY METABOLITES PRODUCED BY YAUPON HOLLY, Ilex vomitoria By Matthew J. Palumbo December 2006 Chair: Francis E. Putz Major Department: Botany Leaves of yaupon ( Ilex vomitoria , Aquifoliaceae), a dioecious shrub native to the coastal plain of the southeastern United States, were traditionally brewed into a caffeinated beverage widely consumed by indigenous peoples and Europ ean colonists. Despite its importance during pre-Colombian and colonial times, yaupon holly is no longer widely exploited for its stimulating chemical properties. Therefore, to foster interest in yaupon, I assessed its commercially harvestable leaf productivity whil e investigating factors that in fluence its production of caffeine and related alkaloids as well as phenolic compounds that partially function as anti-oxidants. First, I tested predictions of the carbonnutrient balance (CNB) hypothesis by determining whether nitrogen availability and plant gender influence prod uction of caffeine and related alkaloids as well as phenolic compounds in leaves of pot-grown yaupon plants fertilized with ammonium nitrate. The CNB hypot hesis predicts that addition al nitrogen should result in increased alkaloid concentrations and decreased phenolic concentrations. An extension of the CNB hypothesis to dioecious plants predicts that females have higher C/N ratios and therefore higher phenolic concentrations and lower alkaloid concentrations than male conspecifics. I found that caffeine and total alkaloid concentrati ons were 5-10 times higher in fertilized than control plants but did not vary by gender. An interaction between ge nder and fertilization,

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10 however, suggests that fe rtilization elicits a grea ter response in caffeine production in females than in males. Total phenolic concentrations were higher in control females than control males as predicted by the CNB hypothesis, but did not vary by treatment; nor were there differences by gender among fertilized plants. In addition, high correlations between antioxidant capacity and both classes of phenolic compounds detected (cinnamic acid deri vatives and flavonoids) indicate that in addition to their putative defensive f unction against herbivores, phenolics protect yaupon from oxidative stress. Results of this study indicate a need to re-appraise the physiological mechanisms by which resource availability affect s secondary metabolism and a need to consider the selective pressures to which secondary metabolism responds. Next, I investigated the su itability of one yaupon cultiv ar (‘Nana’) for commercial production by determining the effects of nitrogen fe rtilization on leaf yield, caffeine content, and antioxidant content. Fertilized plants produced 21.9% more leaf tissue th an control plants and 246.9% higher caffeine concentrations, which resu lted in 350.9% higher total caffeine yield per unit area from fertilized plants. Although antioxi dant capacity on a concentration basis did not differ by treatment, total antioxidant capacity per plant was higher in fertilized plants due to a greater leaf mass yield from fertilized plants. In addition, increases in caffeine concentrations and antioxidant capacity with increasing ca nopy cover suggests that yaupon should be opengrown. Low caffeine concentrations in ‘Nan a’, however, prompted me to assay caffeine concentrations among different ya upon varieties including ‘Nana’. I found that of the commonly cultivated yaupon cultivars in North Florida, ‘Pe ndula’ produces highest and ‘Nana’ the lowest caffeine concentrations. Wild-type varie ties, however, also produced ample caffeine concentrations and may be a bett er choice for sources of cultivars as they provide a wider range of chemical traits, which can be select ed according to the desires of consumers.

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11 CHAPTER 1 NITROGEN FERTILIZER AND GE NDER EFFECTS ON THE SECONDARY METABOLISM OF YAUPON, A CAFFEINECONTAINING NORTH AMERICAN HOLLY Introduction Leaves of yaupon ( Ilex vomitoria , Aquifoliaceae), a dioecious shrub native to the coastal plain of the southeastern United States, were traditionally brewed into a caffeinated beverage widely consumed by indigenous peoples a nd European colonists (Hudson 1979). Although yaupon was the principal ingredient of a beve rage imbibed by Timucuan and Creek Indians during a ceremony that included ritual vomiting (M errill 1979), qualitative chemical analyses detected no emetic properties (Fuller et al. 2002). Partially because of its repellant and misleading specific epithet, yaupon’s virtue as a caffeine source is now mostly ignored, while markets for its close South American relative, Ilex paraguariensis (yerba maté) grow rapidly (Graham 1998). To revive interest in yaupon, we investigated whether nitrogen fertilization increases foliar concentrations of caffeine and ot her methylxanthine alkalo ids. In addition, we determined the effects of nitrog en fertilization on concentratio ns of phenolic compounds, which confer antioxidant properties on ye rba maté (Carini et al. 1998). Finally, we used this dioecious and easily cultivated alkaloid and phenolic-produ cing species to test a well-known theory that addresses nutrient allocation to plant secondary metabolites. The carbon/nutrient balance (CNB ) hypothesis (Bryant et al. 1983; Bryant et al. 1988; Tuomi et al. 1988; Reichardt et al. 1991; Herms and Mattson 1992) is used to explain the effects of nutrient availability on the concentration of plan t secondary metabolites (e.g., alkaloids and phenolics). The CNB hypothesis as serts that plants allocate car bon and nitrogen to secondary metabolism only after growth requirements are me t and that growth is constrained more by nutrient limitations than by photos ynthesis. According to this th eory, the excess carbohydrates that accumulate in nutrient-limited plants when photosynthesis outpaces growth are diverted to

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12 the production of carbon-based secondary comp ounds (e.g., phenolics). Removal of nutrient limitation by fertilization increases tissue nutrient concentrations and allows growth to outpace photosynthesis. Therefore, according to the CNB hypothesis, production of nitrogen-based secondary metabolites (e.g., alkaloids) should in crease as nitrogen is acquired in excess of growth requirements. Conve rsely, concentrations of car bon-based phenolic compounds are predicted to decrease with fe rtilization due to decreased ra tes of photosynthesis relative to growth. Although some studies on the effects of nitr ogen fertilization on secondary metabolites support the CNB hypothesis, others contradict it (Herms and Ma ttson 1992; Koricheva et al. 1998). Critics of the CNB hypothesis explain that its failures can be attrib uted to its assumption that plants respond to varying le vels of resource availability in a physiologically passive manner driven by simple mass-action (Hamilton et al. 20 01; Nitao et al. 2002). These critics contend that although phenotypic plasticity in secondary me tabolite production is influenced by resource availability, it is also subject to selective pres sures and is therefore ge netically regulated and possibly adaptive. For example, plasticity in nicotine production maximizes seed production in Nicotiana attenuata by allowing individuals to regulate resource allocation in response to herbivore pressure (Baldwin 1998; Baldwin 1999). Although variation in production of caffeine a nd phenolics has apparently not yet been shown to be adaptive, there is evidence that their production is gene tically regulated. For example, high narrow-sense heritability for caffeine concentration ( h2 = 0.80) in seeds of robusta coffee ( Caffea canephora ) indicates that most phenotypic va riation in its caffeine output is attributed to genetic and not environmental fact ors (Montagnon et al. 1998) . In other species, phenylalanine ammonia-lyase (PAL) activity, wh ich catalyzes the first step of phenolic

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13 biosynthesis, is finely regulated both by a multigene family and by negative feedback inhibition (Dong et al. 1991; Kervinen et al. 1998). This fine genetic regulation contrasts with the CNB hypothesis that presumes mass flow, as directed by environmental factors, exerts the strongest influence on the production of secondary metabolites (Reichardt et al. 1991). The CNB hypothesis was extended by Herm s and Mattson (1992) to predict gender differences in secondary metabolism. The basis for these predictions is the assumption that due to the nutrient requirements of fruit and seed maturation, female plants allocate higher proportions of nutrients to reproduc tive structures than males. If this assumption is correct, female plants should have higher C/N ratios in vegetative tissue and high er concentrations of carbon-based secondary compounds than males. The logic of this argument notwithstanding, studies examining differences in nitrogen concentration and carbon-based secondary compounds by gender have yielded mixed results (Ågren et al. 1999; Doormann and Skarpe 2002; Bañuelos et al. 2004) indicating that the mechanisms underlying gender differences in secondary metabolism are not yet fully understood. Given the inconsistent evidence for the CNB hypothesis, I sought to contribute to the debate by determining how nitrogen fertiliz ation and gender influe nce the production of methylxanthine alkaloids and phenolics in yaupon holly. In addition, I measured total foliar nitrogen concentrations and an tioxidant capacities to further explore the effects of gender on responses to increased nitrogen av ailability. I hope that while exploring the mechanisms that govern resource allocation to sec ondary metabolism in yaupon, I can stimulate intere st in this long-used but recently forgotten native plant.

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14 Materials and Methods Plant Material and Nitrogen Fertilization Yaupon holly ( Ilex vomitoria ) grows from North Carolina sout h to central Florida and west to east Texas in coastal scrub a nd in the understories of coasta l and inland hardwood forests and pinelands. For this pot study, a rooted leaf-bearing stem was colle cted randomly from each of 26 male and 26 female Ilex vomitoria genets growing in the underst ory of an oak savanna near Gainesville, Florida (29°39´N, 82°19´W). Genets were defined as clusters of stems separated by at least 5 m from the nearest conspecific of th e same gender (as determin ed during the flowering season). Each rooted ramet was planted in a 1 L pot filled with surface soil (0-20 cm) from the collection site. Potted plants were grown outdoors in partial shad e on the University of Florida campus. Percentage full sun for both the source area ( x = .23, s = .16, N = 10) and pot study site ( x= .29, s = .16, N = 4) were similar as estimat ed by Gap Light Anal yzer (GAP) software Version 2.0 (Copyright 1999; Simon Fraser University, British Colombia and Institute of Ecosystem Studies, New York) from photographic images taken with a 180 hemispheric lens (t = .62, P = .55). Although light availability can influence secondary metabolite production (Herms and Mattson 1992, Koricheva et al. 1998), this experiment only allowed assessment of nitrogen fertilization and gender effects. After 8 weeks and continuing for the next 12 week s, half of the plants of each gender were fertilized with ammonium nitrate (NH4NO3) at a rate of 250 mg N per week. Four plants died during cultivation resulting in a final sample size of 13 control males, 10 fertilized males, 12 control females, and 13 fertilized females. All leaves produced after the commencement of the fertilization treatment were harvested for chemical analysis.

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15 Chemical Analyses Collected leaves were dried at 105ºC to a c onstant weight and total nitrogen content was determined using the Kjeldahl method (Bradstree t 1965). Dried leaf tissu e for determination of methylaxanthine alkaloid and phenolic concentra tions was extracted in a 100% methanol for 1 min and filtered through Whatman #4 filter paper. Isolates were then diluted with an equal volume of water, centrifuged for 5 min at ca. 2000 rpm, and filtered through a 0.45 mm filter for HPLC analysis. Separation was conducted with a Waters 2695 Alliance HPLC system using a Supelcosil LC-18 column (250 X 4.6 mm) and de tected at 280 nm with a Waters 996 PDA detector scanned from 200-400 nm. Following the protocol of Talcott et al. (2000) , a gradient mobile phase was run consisting of a 2% aqueous solution of acetic acid in phase A and a 30% acetonitrile plus 2% acetic acid solution in phase B. The gradient ran phase B from 0-30% for 20 min, 30-50% for 10 min, 5070% for 20 min, and 70-100% for 5 min at 0.8 mL/mi n. Phase B ran for an additional 15 min to elute remaining non-polar compounds followed by e quilibration of the column with 100% phase A prior to injection of the ne xt sample. Compounds were identified by UV/VIS spectral interpretation and compared to authentic standard s and retention times when available. Alkaloid and phenolic concentrations were calcu lated on percent dry weight basis. Isolates extracted in methanol were also used to quantify total reducing compounds by the Folin-Ciocalteu method (Swain and Hillis 1959), which provides an estimate of total soluble phenolics. Data were expressed in chlorogenic acid equivalents. Th e same isolates were used to determine antioxidant capacity by the oxygen radical absorbance capacity (ORAC) method as modified by Ou et al. (2001). Fractions were diluted 400-fold in pH 7.2 phosphate buffer prior to pipetting into a 96 well microplate. Peroxyl radicals were generated by 2, 2’-Azobis (2amidinopropane) dihydrochloride (AAPH) with fluorescein as the fluorescent probe.

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16 Fluorescence loss was measured on a Molecular Devices fmax Microplate Reader (485 nm excitation, 538 nm emission, 37°C) every 2 min for 70 min. Areas under decay curves generated by these measurements were compared to areas fo r blank preparations and a standard curve of Trolox, a water-soluble analogue of toc opherol (vitamin E) and expressed as Trolox equivalents/g. Statistical Analyses Differences in chemical concentrations betw een treatments were analyzed with a 2-way full factorial ANOVA followed by Tukey’s HSD tests for post-hoc pair-wise contrasts. Correlations were determined by Pearson product-moment correlation. Results Methylxanthine Alkaloids Concentrations of caffeine, theobromine, a nd total methylxanthine alkaloids (Figure1-1) were substantially higher in leaves of fertiliz ed than control plants (Table 1-1). Although concentrations of these compounds did not vary by gender, there was a significant interaction between fertilization and gender in the caffeine response (Table 11). This interaction may be explained by a greater response to fertilization in females (10.4-fold increase) than males (5.6fold increase). In contrast to the caffeine effect , there were no significant interactions in either the theobromine or total methyl xanthine alkaloid responses. Phenolics Concentrations of total solubl e phenolics in yaupon foliage did not differ by treatment or by gender but were influenced by an interaction be tween the two effects (Table 1-2). Post-hoc analysis (Figure 1-2) indicates that the interaction is at least partia lly attributed to slightly higher total phenolic concentrations in control females than control males ( P < .05).

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17 HPLC analysis of yaupon leaf issue detected two classes of pheno lic compounds, cinnamic acid derivatives and flavonoids. Based on retent ion times, maximum wavelength of absorbance, and UV spectroscopic analysis by PDA, the cinnamic acid derivatives were composed of chlorogenic acid, chlorogenic acid isomers, and chlorogenic acid di-isomers (dicaffeoyl-quinic acids). Based on the above indicators as we ll as a PDA-library of flavonoid samples, the flavonoids are most likely rutin and luteolin. Concentrations of cinnamic acid derivatives, total flavonoids, and total phenolics as detected by HPLC did not vary by gender or in response to fertilization (Table 1-2) but these treatments interacted to aff ect the production of cinnamic acids and total phenolics detected by HPLC (Table 1-2). As found with total soluble ph enolics, post-hoc pair-w ise tests suggest that these interactions are attributable to greater c oncentrations of both cinna mic acid derivatives and total HPLC phenolics in control females than control males ( P < .05). Fertilized samples, in contrast, differed neither by gende r nor from control plants in concentrations of cinnamic acid derivatives or total HPLC phenolics and there wa s no gender-by-fertilizati on interaction in the flavonoid response. Nitrogen Content Although foliar nitrogen concentrations (Figure 11) were much higher in fertilized than control plants ( F = 233.2, P < .0001), nitrogen concentration di d not vary by gender nor were there significant interactions. In all plants, nitr ogen concentrations were closely correlated with concentrations of caffeine ( r = .98, P < .0001), theobromine ( r = .67, P < .0001), and total methylxanthine alkaloids ( r = .98, P < .0001). Furthermore, a far greater proportion of nitrogen was invested in alkaloid producti on by fertilized plants. For exam ple, female and male control plants invested 7.4 and 12.8 % of their total ni trogen in the production of methylxanthine

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18 alkaloids, respectively, whereas in fertilized females and males, the respective proportions were 29.8 and 28.3%. Antioxidant Capacity Measures of oxygen radical absorption capacity (ORAC) were extremely high in all plants and did not vary by treatment or gender (Figure 1-2). ORAC values were highly correlated with total phenolics ( r = .94, P < .0001), total cinnamic acid derivatives ( r = .93, P < 0.0001), total flavonoids ( r = .58, P < .0001), and total phenolics as measured by HPLC ( r = .93, P < .0001). Discussion The results of this study both support and c ontradict predictions of the CNB hypothesis. Yaupon responded to nitrogen fertiliz ation with large increases in concentrations of caffeine and total methylxanthine alkaloids but not with de creases in concentrations of cinnamic acid derivatives, flavonoids, or total phenolics. Furthermore, neither alkaloid nor total nitrogen concentrations differed by gender, although an interaction between gender and fertilization influenced caffeine production. As predicted by the CNB hypothesis, cinnamic acid derivative and total phenolic concentrations were higher in females than males among the control group but did not differ by gender among fertilized plants no r did flavonoid concentr ations differ by gender in either control or fertilized groups. The lack of phenolic responses by yaupon to nitrogen addition in yaupon indicates the need for alternatives to the mechanism set fo rth by the CNB hypothesis for interpretation of these results. Although the CNB hypothesis pred icts that carbon allo cation to carbon-based secondary metabolism decreases with nitrogen fertilizati on due to faster rates of growth relative to photosynthesis (Bryant et al. 1983), nitrogen fertilization does not necessarily favor growth over photosynthesis (Kannan and Paliwal 1997). Fu rthermore, even when nitrogen additions stimulate biomass accumulation but not photosynth etic rates, phenolic concentrations do not

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19 always decrease (Reichardt et al. 1991; Bezemer et al. 2000). Similarly, Donaldson et al. (2005) found no correlation between relativ e growth rates and the rela tive foliar mass of phenolic glycosides among n itrogen-fertilized Populus tremuloides . The CNB hypothesis places an importan ce on relative increase s of growth over photosynthesis in response to nitrogen fertiliza tion because it posits that secondary metabolite production is driven by carbon and nitrogen overflow beyond allocati on to growth (Bryant et al. 1983; Reichardt et al. 1991). By this mechanis m, relatively lower photos ynthetic rates with additional nitrogen constrain ca rbohydrate availabili ty for carbon-based secondary metabolism while nitrogen supplied in excess of growth re quirements is allocate d to nitrogen-based secondary metabolism. Studies have revealed, ho wever, that nitrogen fertilization does not necessarily decrease starch or total sugar concentrations ev en when it decreases phenolic concentrations (Balsberg PÃ¥hlsson 1992) and th at nitrogen additions may even stimulate increases in concentrations of total nonstructu ral carbohydrates (Kaakeh et al. 1992). Closer examination of the influence that ca rbohydrate metabolism exerts on all secondary metabolic pathways may resolve patterns of resource allocation to secondary metabolite production. In particular, biosynthesis of nitr ogen-based secondary compounds is dependent on carbon skeletons generated by photos ynthesis (Dennis and Blakeley 2000) and therefore, like the biosynthesis of carbon-based secondary co mpounds, should also be limited by carbon availability. For example, biosynthesis of phe nylalanine, the amino acid precursor of phenolic compounds, is dependent on organic acids de rived from carbohydrate pools generated by glycolysis and the pentose phosphate pathwa y (Weaver and Herrmann 1997). Similarly, biosynthesis of purine, the nucleo tide precursor of caffeine and re lated methylaxanthine alkaloids (Suzuki et al. 1992), also begins with an or ganic acid derived from the pentose phosphate

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20 pathway and derives nitrogen from amino acids with carbon skeletons also supplied by organic acids (van der Graaff et al. 2004) . Carbon partitioning obviously warrants further investigation in yaupon and other plant species that empl oy numerous secondary metabolic pathways. A refined understanding of the mechanisms by which nitrogen availability influences carbohydrate, nitrogen, and secondary metabolism can clarify its influence on carbon and nitrogen allocation for the production of both al kaloids and phenolics. For example, recent studies have shown that nitrate, and not mere ly downstream nitrogen metabolites, signals the induction of organic acid, amino acid, and nuc leotide metabolism but represses starch biosynthesis (Scheible et al. 1997; Scheible et al. 2004). By a si milar mechanism, nitrate also induces nicotine but inhibits phenylp ropanoid and flavonoi d biosynthesis in Nicotiana tabacum (Fritz et al. 2006). These studi es indicate that carbohydrate, n itrogen, and secondary metabolism are, in fact, coordinated according to nitrogen av ailability. C/N ratios alone, however, may be insufficient for predicting resour ce allocation to secondary me tabolites because their production is influenced by nitrate-induced signals and not merely by the mass flow of carbon and nitrogen through various metabolic pathwa ys (Stitt and Krapp 1999). Root-shoot ratios are also known to reflect C/ N ratios (e.g., Ã…gren and Ingstead 1987) and therefore may shape the effect of nitrogen a dditions on carbohydrate, nitrogen, and secondary metabolic responses. For example, studies that showed a decrease in root/shoot ratios with additional nitrogen also showed concomitant increases in soluble sugar and amino acid concentrations but decreases in starch concentr ations (Green et al. 1994 ) as well as decreased concentrations of condensed tann ins but no changes in benzoic acid derivatives (Donaldson et al. 2006). Because our study on ya upon involved the use of rooted vegetative offshoots with

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21 presumably low root/shoot ratios (e.g., Ritchie et al. 1992), the potential infl uence of root/shoot ratios on secondary metabolic ou tput should be considered. Determining the influence of selective agen ts may also inform our understanding of the physiological mechanisms that drive variation in secondary metabolic output (Berenbaum 1995; Hamilton et al. 2001; Nitao et al. 2002). For example, give n that many plant secondary metabolites are putative defenses against pathogens and herbivores, predictions about allocation patterns for their production should reflect the se lective pressures to wh ich they are a response (Berenbaum 1995). Induction of caffeine and/or theobromine synthesis upon attack by fungal pathogens in tea ( Camellia sinensis ; Kumar et al. 1995; Punyasiri et al. 2005) and cacao ( Theobroma cacao ; Aneja and Gianfagna 2001), for example, provides evidence that selective agents may shape the production patterns of these alkaloids. Although plant nutrient concentrations have long been correlated w ith herbivore preferences (e.g., Mattson 1980), the effect of nitrogen availability on chemical responses to herbivor es and pathogens has not been extensively explored. Lou and Baldwin (2004) found, however, that plants with low nitrogen availability maintain higher constitutive levels of nicotine than plants with high nitrogen availability but that only plants of the latter group increased ni cotine production in response to application with larval oral secretions in Nicotiana attenuata . Secondary metabolites may serve adaptive func tions in addition to defense, which may also help explain their respons es to additional nitrogen (Herms and Mattson 1992; Nitao et al; 2002). For example, our results indicating that fertilized yaupon plants allocated a higher proportion of nitrogen to methylxa nthine alkaloid production than control plants suggest that alkaloid biosynthesis is not only actively regulated in yaupon ho lly but that caffeine may also serve multiple functions. Caffeine concentrations in fertilized female and male plants (3.9% and

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22 3.4%, respectively) far exceed those necessary to deter or even kill herbivores (Nathanson 1984; Slansky and Wheeler 1992, Hollingsworth et al. 2003). Their high allocation of nitrogen to caffeine after nitrogen fertilizati on suggests that caffeine also serves as a storage metabolite for nitrogen accumulated in excess of immediate plant needs (Chapin et al. 1990). The suitability of caffeine for nitrogen storage, however, require s verification by studies addressing both the metabolic and ecological costs of storing caffeine. The multiple functions of phenolic compounds may render it difficult to predict their production in response to nitrogen additions as well. For example, chlorogenic acid and flavonoids are known to inhibit herbivores (e.g., Ikonen et al. 2001; Ikonen et al. 2002: Onyilagha et al. 2004) but both also protect plants from light stre ss by scavenging free radicals and absorbing potentially harmful UV radiati on (Grace and Logan 2000; Harborne and Williams 2000). Correlations between antioxidant capacity and all measures of phenolic concentrations indicate that both cinnamic acid derivatives and flavonoids provide yaupon with protection from oxidative stress. That a particular secondary metabolite serves numerous functions supports the notion that its production is under genetic cont rol and most likely regulated by numerous stress factors (Nitao et al. 2002), making it difficult to ascertain the mechanisms driving its phenotypic variation without first isolating the effects of each factor. Our results also largely contra dict the CNB hypothesis predicti on that female plants have higher foliar C/N ratios, lower levels of nitr ogen-based defensive compounds, and higher levels of carbon-based defenses than male conspecifics (Herms and Mattson 1992; Ã…gren et al 1999). In particular, concentrations of neither alkalo ids nor total nitrogen di ffered by gender in yaupon. The observed interactive effect between ge nder and fertilization on caffeine production, however, suggests that female plants respond more to nitrogen fertiliz ation than males, and

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23 therefore that resource allocation to secondary metabolism may differ by gender. Furthermore, higher concentrations of total phenolics and total cinnamic acid de rivatives in control females than control males also suggest gender diffe rences in resource allocation to secondary metabolism. Measurements of other traits by gender such as growth rates, herbivory, and investment in structur al defenses (Jing and Coley 1990; Ã…g ren et al 1999) may resolve the mechanism and selective forces that shape vari ation in secondary metabolic output. If genderbased phenotypic differences in life history traits are obser ved, however, they should be appraised in terms of their effects on reproduc tive success or some other measure of their adaptive significance (Berenbaum 1995). The dramatic increases in caffeine and th eobromine concentrati ons in response to increased nitrogen availability observed in ya upon may relate to its success across a broad environmental range (Sultan 2000). For exam ple, yaupon occurs both in coastal scrub communities, where it is adap ted to high light, salt spray, and drought (Johnson and Barbour 1990), as well as in the understori es of mesic hardwood forests, which are typically moister and contain higher levels of soil organic matter (Pla tt and Schwartz 1990). Because foliar nitrogen concentrations affect plant susceptibility to herbivory (Mattson 1980) and because the native habitats of yaupon vary in soil nutrient availabi lity, an ability to me diate between herbivore pressure and defensive metabolite production in re sponse to nitrogen availability may indicate a plastic response that is possibly adaptive. Su ch an adaptive strategy may be suggested by our finding that 2.4and 2.3-fold increases in foliar nitrogen concentration resulted in 10.4and 5.6fold increases in the caffeine concentrations of female and male plants, respectively. Future studies that include the identification of ya uponÂ’s herbivores and pathogens may reveal the significance of these differences in allocation to caffeine in relative propor tion to total nitrogen

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24 concentration. Common garden a nd reciprocal transplant experime nts could also help to clarify the role that phenotypic plasticity in secondary metabolic output pl ays in determining the success of yaupon across its large geograp hical and ecological range. Conclusions Because yaupon holly ( Ilex vomitoria ) is dioecious, easily cu ltivated and produces large amounts of both nitrogen-based and carbon-base d secondary compounds, it is an excellent species on which to test theories about reso urce allocation to secondary metabolism. For example, although an increase in methylxanthine al kaloid concentrations in response to nitrogen fertilization is predicted by the CNB hypothesis, the lack of pheno lic responses i ndicates that a re-evaluation of the mechanism by which nitrogen availability in fluences secondary metabolism is necessary. In addition, the lack of differences in concentrations of both total foliar nitrogen and methylxanthine alkaloids between genders co ntradicts an extension of the CNB hypothesis to dioecious species. An in teractive effect between gende r and fertilization on caffeine production as well as higher phenolic concentrati ons in female conspecifics, however, indicates that resource allocation to s econdary metabolite production may differ by gender in yaupon. The precise mechanisms underlying these gender differen ces, however, warrant fu rther investigation. Finally, ample production of both caffeine and an tioxidant-rich phenolics indicate the potential for renewed commercial use of yaupon holly.

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25 Table 1-1. ANOVA results for foliar concentratio ns of methylxanthine (MX) alkaloids in Ilex vomitoria . Total Source df Caffeine Theobromine MX Alkaloids Treatment 1 329.04*** 88.29*** 348.89*** Gender 1 0.47 0.79 0.25 Treatment*Gender 1 4.26* 0.18 3.55† Error 44 F values presented for main effects and their in teraction. Levels of significance indicated as follows: * P < 0.05, *** P < 0.0001, † P < 0.1. Table 1-2. ANOVA results for foliar con centrations of phenolic compounds in Ilex vomitoria . Total Total Total Total Source df Phenolics Cinnamic Flavonoids Phenolics Derivatives by HPLC Treatment 1 1.83 1.67 0.58 1.65 Gender 1 0.19 0.13 3.48† 0.22 Treatment*Gender 1 6.21* 6.10* 2.84† 6.12* Error 44 F values presented for main effects and their in teraction. Levels of significance indicated as follows: * P < 0.05, † P < 0.1.

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26 Figure 1-1. Mean (+1 SE) foliar concentrations of alkaloids and ni trogen by treatment and gender in Ilex vomitoria (note difference in scales). A) Caffeine. B) Theobromine. C) Total methylxanthine alkaloids. D) Total ni trogen. Black bars represent female plants; grey bars represent male plants. Lower case letters specify groupings as indicated by TukeyÂ’s Tests for multiple comparisons when interactions were indicated ( P < .05).

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27 Figure 1-2. Mean (+1 SE) foliar concentratio ns of phenolics and antioxidant capacity by treatment and gender in Ilex vomitoria (note difference in scales). A) Total soluble phenolics. B) Cinnamic acid derivatives. C) Flavonoids D) Total HPLC phenolics. E) Antioxidant capacity. Black bars represent fe male plants; grey bars represent male plants. Lower case letters specify gro upings as indicated by TukeyÂ’s Test for multiple comparisons when interactions were indicated ( P < .05).

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28 CHAPTER 2 Ilex vomitoria : AN OVERLOOKED NORTH AMERICAN CAFFEINE SOURCE Introduction Caffeine and Conservation Caffeine, the world’s most highly consumed psychoactive substance (Weinberg and Bealer 2001), affects numerous socio-economic issues at a global scale. For example, when production of sun-grown coffee in Southeast Asia increas ed in the 1990s while world consumption rates remained constant, its market value crashed (Vandemeer 2003). This ‘global coffee crisis’ triggered a positive feedback whereby increased areas of forest were cleared for coffee production to compensate for financial losses, wh ich in turn threatened the conservation of tropical Asian megafauna including Sumatran tige rs, elephants, and rhin oceroses (O’Brien and Kinnaird 2003). Although solutions to this crisis like improvi ng yields of sun-grown coffee while discouraging its cultivation in protected wildlife areas have been offered and debated (O’Brian and Kinnaird 2004, Dietsc h et al. 2004), we suggest that local sources of caffeine be exploited to alleviate pressure on forest conversion in areas of hi gh conservation value. In North America, for example, consumers might consid er replacing coffee with yaupon holly for their stimulating morning beverage. Yaupon ( Ilex vomitoria ), a caffeine-containing shrub that was long used in its native ra nge of the southeastern United States but recently forgotten, is the focus of the following study, which exam ines several factor s associated with its production as a caffeine crop. History of Yaupon Tea Consumption of a tea made from yaupon holly began with the indigenous peoples of its native range who were presumably the first to discover its stimulating and healthful properties (Hudson 1979). Amerindians brewed leaves and twigs of yaupon holly into a caffeine-

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29 containing beverage whose cons umption figured prominently in their social and cosmologic constructs (Hudson 1995). For example, consump tion of tea made from yaupon in Creek society was reportedly reserved for adult males of hi gh social status and was associated with Yahola , a sky deity revered for his immaculate nature (M erill 1979, Fairbanks 1979). Rituals that included fasting, consumption of large quantities of yaupon tea, and vomiting reflect the importance that these peoples placed upon spiritual purity and cl eanliness deemed necessary before assembling for important council meetings or military act ion. The act of vom iting was more likely a manifestation of a symbolic connection between yaupon and purity than a physiological response to ingestion of yaupon tea, which was consumed in settings that did no t include vomiting (Merill 1979) and does not seem to possess any chemical properties that would i nduce emesis (Fuller et al. 2002). European settlers of the sout heastern United States readily adopted the Amerindian practice of brewing and dr inking yaupon tea. Spanish settlers in Florida, for example, learned of its virtues from the Timucua Indians in the 16 th Century (Hudson 1979). Yaupon tea became so popular among Spanish colonists, who called it te del indio or cacina (Hudson 1979, Hudson 1995), that a Spanish priest reported in 1615 that “there is no Spaniard or Indian who does not drink it every day in the morni ng or evening” (Sturtevant 1979). By the 18th Century English settlers in South Carolina also began drinking yaupon tea, which they called “black drink” or cassina in South Carolina (Hudson 1979, Hudson 1995) and yaupon in North Carolina (Hale 1891). Europeans presumably found the taste of yaupon tea agreeable as it appeared in 18th Century markets in both England, where it wa s called Appalachian tea or Carolina tea, and France, where it was called Appalachina (Hudson 1979). Despite its initial acceptance in

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30 Europe, broad consumption of yaupon tea ceased bot h in the United States and abroad by the late 19th Century (Hudson 1979, Hudson 1995, Sturtevant 1979). The disappearance of yaupon tea from common us e has long puzzled scholars especially given that caffeine is the world’s most popular drug and caffeinated beverages are in high demand (Hale 1891, Hudson 1979, Sturtevant 1979). Hale (1891) suggested that diminished consumption of yaupon might be attributed to a l ack of cultural exchange between the European settlers and indigenous peoples of the Southeastern United States. This lack of social cohesion contrasts with the mixing of races that occurred in South America, home of yerba maté , a caffeinated beverage prepar ed from a congener of yaupon, Ilex paraguariensis . Yerba maté has maintained its popularity to the present era. Hudson (1979), in contrast, speculated that discontinued use of yaupon is attri buted to interference of its tr ade by tea and coffee merchants, which led to its association w ith an underclass that could no t otherwise afford the more expensive, aforementioned caffeine-containing products. Although differences in several key socio-economic factors may exist in the consumption of yaupon tea and yerba maté , historical uses of these beverages are otherwise very similar. European consumption of yerba maté began circa 1540 when Spanish colonists adopted it from the Guaraní Indians and spread from its native range of southern Brazil, Paraguay, Uruguay, and northeastern Argentina to Andean c ountries as far north as Ecuador by the 18th Century (Sturtevant 1979, Jamieson 2001). When coffee and tea became the caffeinatedbeverages of choice by South America’s uppe r classes in the 19 th century, however, yerba maté was relegated to status as the drink of th e lower classes (Jamieson 2001). Nevertheless, consumption of yerba maté in South America managed to pe rsist alongside other caffeinated beverages in the 20th Century perhaps because it maintain ed regional identity in the face of

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31 increasingly globalized markets (J amieson 2001). Annual production of yerba maté in Argentina, Paraguay, and Uruguay currently exceeds 250,000 Mg (Graham 1998). Furthermore, yerba maté has recently become a global commodity, as evidenced by a nearly 14-fold increase in revenue generated from Argentinean e xports between 2002 and 2004 (United Nations Commodity Trade Statistics Database). This gl obal commercialization has been bolstered by the reportedly high antioxidant content and a ssociated human health benefits of yerba maté (Bastos et al. 2006) as similarly reputed for green tea prepared from Camellia sinensis (Stewart et al. 2005). Yaupon Chemistry and Production Many Americans might be surprised to learn of a native caffeine-pr oducing plant that can be used to brew a yerba maté -like beverage. A bigger surprise may come when residents of the Southeastern United States learn that they ofte n encounter this species and may even have it growing it in their backyard. Ya upon is widely cultivated as an ornamental hedge on urban and suburban landscapes within its native range and at least 17 cultivars are currently marketed (Bijan Dehgan, personal communication). Ea rly endorsements of yaupon cultivation for commercial exploitation of its chemical prop erties (Hale 1891, Power and Chestnut 1919), however, were mostly ignored. In my previous study of the chemistry of ya upon (Chapter 1), I found that fertilization of pot-grown plants with nitrogen substantially in creases foliar concentrations of caffeine and theobromine. In addition, I found that yaupon foli age possesses a high anti oxidant capacity that is tightly correlated with the production of phenolic compounds but that is unaffected by additional nitrogen. That study reve aled that appraisal of several other factors critical to the production of yaupon was still necessary. For exam ple, given the availability of numerous cultivars, it seems important to sc reen them so as to identify t hose with desired levels of both

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32 caffeine and antioxidants. Similarly, leaf producti on and thus yields of caffeine and antioxidants from fertilized and unfertilized plants are unkno wn. Finally, the influence of canopy cover on the production of both alkaloids and antioxidants in yaupon should be considered because one popular plant defense theory, the carbon/nutrien t balance (CNB) hypothesis, predicts that exposure to increased light incr eases production of antioxidant-ri ch phenolics while decreasing alkaloids like caffeine (Bryant et al. 1983) Because antioxidants have reputed health be nefits, it may be important to the economic value of yaupon that high antioxidant levels be maintained. The health benefits of antioxidants are partially attributed to thei r ability to prevent the formation, scavenge, or promote the decomposition of free-radicals, which are know n to contribute to numerous human health problems such as cancer, cardiovascular diseas e, diminished immune system function, brain dysfunction, and cataracts (Ames et al. 1993, Young and Woodside 2001). For example, prevention of lipid peroxidati on and DNA fragmentation caused by free radical formation, which are known to cause cardiovascular disease and cancer, respectively, has been attributed to antioxidants present in both yerba maté (e.g., Schinella et al. 2000, Bracesco et al. 2003) and green tea (e.g., Terao et al. 1994, Yokozawa et al. 1998). In the following study, I investigated the suitability of one yaupon cultivar for commercial production of leaves for tea by determining its leaf production rates, foliar caffeine concentrations, and foliar antioxidant capacity wi th and without nitrogen fertilization. In this cultivar, I also evaluated the in fluence of light availability on the production of both caffeine and antioxidants. Finally, I compar ed caffeine concentrations among three cultivars of yaupon and wild-type yaupon in order to de termine which yaupon varieties are best suited for commercial production of foliage for brewing tea.

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33 Materials and Methods Experimental Design Yaupon shrubs comprising six separate hedges of the ‘Nana’ cultivar (Figure 2-1) were sampled on the University of Florida (UF) campus in Gainesville, Florida (29°39´N, 82°19´W). Leaves covering an area of 700 cm2 were clipped from the prune d upper surface of two shrubs per hedge. For 3 months during the growing se ason, one shrub per hedge was then fertilized monthly with 250 mg ammonium nitrate (NH4NO3) at a rate of 1g fertilizer/m2/mo; the other shrub in the same hedge but separated by >2 m was reserved as an unfertiliz ed control. After 3 months, leaves were clipped from the same area as the original clipping. Leaves from the second harvest were then dried at 60 û C for 48 hours and weighed after which 0.5 g of dried and ground leaf material was extracted with water in test tubes that were then placed in boiling water for 10 minutes. Extracts were then analyzed for caffe ine concentration and antioxidant capacity using the methods described in Chapter 1. To compare caffeine concentrations in the foliage of different yaupon varieties, nine separate shrubs each from three cultivars of yaupon (‘Nana’, ‘Pendul a’, and “Will Fleming’) were randomly sampled from cultivated hedges on the UF campus. In addition, leaves were collected from five wild-type yaupon shrubs occu rring in natural stands on UF campus and four wild-type shrubs growin g in an oak savanna approximately 4 km away. The youngest, fully expanded leaves were harvested from each of the four varieties and processed as above. Chemical Analyses To determine caffeine concentrations in yaupon foliage, the water extracts were centrifuged for 5 min and filtered through a 0.45 mm filter for HPLC analysis. Separation was conducted with a Waters 2695 Alliance HPLC syst em using a Supelcosil LC-18 column (250 X 4.6 mm) and detected at 280 nm with a Waters 996 PDA detector scanned from 200-400 nm.

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34 Following the protocol of Talcott et al. (2000), a gradient mobile phase was run consisting of a 2% aqueous solution of acetic acid in phase A and a 30% ace tonitrile plus 2% acetic acid solution in phase B. The gradient ran phase B from 0-30% for 20 min, 30-50% for 10 min, 5070% for 20 min, and 70-100% for 5 min at 0.8 mL/mi n. Phase B ran for an additional 15 min to elute remaining non-polar compounds followed by e quilibration of the column with 100% phase A prior to injection of the next sample. Caffeine was identified by UV/VIS spectral interpretation and compared to a caffeine sta ndard solution. Caffeine concentrations were calculated on percent dry weight basis. The isolates used to determine caffeine c oncentration were also used to measure antioxidant capacity by the oxygen radical absorban ce capacity (ORAC). Fractions were diluted 400-fold in pH 7.2 phosphate buffer prior to pi petting into a 96-well microplate. Peroxyl radicals were generated by 2, 2’-Azobis (2 -amidinopropane) dihydrochl oride (AAPH) with fluorescein as the fluorescent pr obe. Fluorescence loss was m easured on a Molecular Devices fmax Microplate Reader (485 nm excitation, 538 nm emission, 37°C) every 2 min for 70 min. Areas under decay curves generated by these meas urements were compared to areas for blank preparations and a standard curve of Trolox, a water-soluble analogue of tocopherol (vitamin E) and expressed as Trolox equivalents/g. The water extracts derived from the sec ond study comparing caffeine concentrations among yaupon varieties were centrifuged for 20 minutes and filtered through a 0.45 mm filter for HPLC analysis. Separation was conducted with a Waters 717 Plus Autosampler HPLC system using a Dionex C-18 column ( 250 X 4.6 mm) and detected at 280 nm with a Waters Dual Absorbance Detector scanned. Following a modified protocol of Talcott et al. (2000), a gradient mobile phase was run consisting of a 2% aqueous solution of acetic acid in phase A and a 30%

PAGE 35

35 acetonitrile plus 2% acetic acid solution in phase B. The gradient ran phase B from 0-30% for 20 min, 30-50% for 10 min, 50-70% for 3 min, a nd 70-100% for 2 min at 1.0 mL/min. Phase B ran for an additional 5 min to elute remaining non-polar compounds followed by equilibration of the column with 100% phase A prior to inj ection of the next sample. Compounds were identified by comparison of spectra and rete ntion time with a caffeine standard solution. Caffeine concentrations were calcula ted on percent dry weight basis. Measurement of Light Availability Percentage full sun for each of the six yaupon hedges sampled in the nitrogen fertilization study was estimated by Gap Light Analyzer (GAP) software Vers ion 2.0 (Copyright 1999; Simon Fraser University, British Colombia and In stitute of Ecosystem Studies, New York) from photographic images taken with a 180 hemispheric lens. Statistical Analysis Caffeine content and antioxidant capacity fo r each sample of the nitrogen fertilization study were calculated by multiplying by total leaf mass. Differences in total leaf mass, caffeine concentration, total caffeine content, antioxidant capacity, and total antioxidant capacity content were then analyzed by paired-sample t-tests. Effects of canopy cover on caffeine concentrations and antioxidant capacity were determined by linear regressions. Caffeine concentrations in the foliage of the three cultivars and wild -type yaupon were compared by one-way ANOVA followed by Tukey’s HSD test for po st-hoc pair-wise contrasts. Results Fertilized plants of the ‘Nana’ variety of I. vomitoria yielded only 1.2% more leaf tissue (t = 2.95, N = 6, P < 0.05) than control plants over the 3 month period monitored but the foliage produced contained 3.5% higher caffein e concentrations (t = 3.16, N = 6, P < 0.05), which resulted in 4.5% higher total caffeine yiel d from fertilized plants (t = 2.69, N = 6, P < 0.05;

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36 Figure 2-2). Although antioxidant capacity on a concentration ba sis did not differ by treatment (t = 1.22. N = 6, P = 0.14), total antioxidant capacity per plant was higher in fertilized plants (t = 2.07, N = 6, P < 0.05) due to their total l eaf mass yield (Figure 2-3). Measurements of canopy cover revealed a pos itive influence of light availability on the production of both caffeine and antioxidants. A marginally significant relationship between caffeine concentration and percen tage full sun was detected [caffeine concentration = -0.11 + 0.009 (% full sun); SEb = 0.005; P = 0.1; R2 = 24.7; Figure 2-4] sugge sting a positive influence of light availability on caffeine production. The effect of light on ORAC was stronger; antioxidant capacity increased ra pidly and consistently with increasing light levels [oxygen radical absorbance capacity = 210.4 + 16.0 (percentage full sun); SEb = 3.2; R2 = 72.1; P < 0.001; Figure 2-5]. Caffeine concentrations varied among the among yaupon varieties sampled in the second part of this study ( F = 3.13, P < 0.05). Post-hoc pair-wise co mparisons indicate that foliar caffeine concentrations were highest in the ‘P endula’ variety, lowest in variety ‘Nana ’ and intermediate in “Will Fleming’ and wild-type yaupon (Figure 2-6). Discussion Yields of caffeine and antioxidant capacity in yaupon hedges, esp ecially after nitrogen fertilization, suggest th e suitability of yaupon for producing a s timulating and healthful beverage. The caffeine concentrations in both the control and N-fertilized ‘Nana’ cultivar hedges were disappointingly low (Figure 2-2) compared with other studies on wild-type individuals. For example, in a study that assayed leaves harv ested from individuals of yaupon growing in maritime forest, caffeine concentrations ranged from 0.35 to 0.94% (Edwards and Bennett 2005).

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37 Similarly, in the pot study described in Chapter 1, control plants range d 0 to 1.91% caffeine and one fertilized plant had a caffeine concentration of 5.25% caffeine. Although measures of antioxidant capacity in ‘Nana’ were moderately high, as measured by the ORAC method, they were lower than those found in my previous study of yaupon (Chapter 1), as well as in Asian green tea and yerba maté (Chandra and Gonzalez de Mejia 2004; Table 1). Such comparisons need to be ma de with care because of the strong positive relationship I found between light availability and antioxidant production. This finding also suggests that if antioxidants are favored by cons umers, yaupon should be grown at high light intensities. Although there was only a weak positive relationship between light availability and caffeine production, it still appears that hi gh light conditions may increase caffeine concentrations in yaupon, which is contrary to a prediction of th e carbon/nutrient balance (CNB) hypothesis (Bryant et al. 1983). Comparison of caffeine concentrations among yaupon varieties indicat es that ‘Pendula’ may be the best candidate for cultivati on as a caffeine crop among common cultivars. Development of cultivars from wild-type varietie s with desired caffeine concentrations, however, may be a better option as indicated by these re sults and those from st udies mentioned earlier (Chapter1, Edwards and Bennett 2005), which showed a wide range of caffeine concentrations. The extent to which this variation is genetically based has yet to be determined, but it is clear that yaupon cultivar ‘Nana’ is not the best choice for cultivation if caffeine production is to be favored. The appropriateness of yaupon as a caffeine sour ce is also indicated by its ample caffeine concentrations, which are comparable to those found in unprocessed leaves of yerba maté whose reported caffeine concentrations vary from 0.65 0.85% (Mazzaferra 1994, Reginatto et al.

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38 1999). In future studies, the influence of methods employed in both the processing and preparation of caffeinated beverages should be addressed. For example, more caffeine is apparently lost in the processing of yerba maté by the barbaqua method than by the supelco method due to the higher temperatures to which le aves are exposed in the former (Graham 1998). The relationship between the caffeine concentrat ion of the caffeinated product and amount of product used in the preparation of beverage al so warrants consideration. For example, although Asian tea leaves from Camellia sinensis contain ~ 3.5% caffeine and coffee beans derived from Coffea arabica contain ~1.1% caffeine by we ight (Spiller 1984), a 177 cm3 cup of tea contains 25 100 mg of caffeine as compared to the 130 – 180 mg of caffeine found in the same volume of Arabica coffee due to the higher mass of product used in its preparation (Weinberg and Bealer 2002). Finally, the method in which a caffeinated beverage is prepared greatly influences its caffeine concentration. For example, coffee prep ared by the drip method contains more caffeine than that prepared by percolation, which again co ntains more caffeine than instant coffee (Gilbert 1986). Conclusions Yaupon holly ( Ilex vomitoria ) provides North Americans with the opportunity to secure a native caffeine source and therefore mitigate th e socio-economic and biological conservation problems associated with the consumption of othe r caffeinated beverages. Yaupon is capable of producing ample concentrations and yields of both caffeine a nd antioxidants, which can be adjusted to desired levels thr ough alteration of soil nutrient and light regimes. The value of yaupon is increased by its high antioxidant capacit y, which has bolstered the value of its South American congener yerba maté ( Ilex paraguariensis ), due to the reported health benefits it provides. Although this study indicates yaupon cultivar ‘Pendula’, currently used as an ornamental hedge, produces caffe ine concentrations on par with yerba maté , I suggest the use of

PAGE 39

39 wild-type varieties in the deve lopment of cultivars for tea production because they offer a wider range of chemical traits that can be selected ac cording to the desires of consumers. Finally, I hope that this study inspires people living in the range of yaupon to forage for its leaves and to prepare a stimulating and healthful drink that will link them to the histor y of the region.

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40 Figure 2-1. Cultivated hedge of yaupon ( Ilex vomitoria ) variety ‘Nana’ on the University of Florida campus, Gaines ville, Florida.

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41 Figure 2-2. Mean (+/SE) A) leaf mass, B) caffe ine concentration, and C) total caffeine yield in control and fertilized Ilex vomitoria cultivar ‘Nana’ individuals obtained from the second harvest of this study (analysis based on paired samples, N=6).

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42 Figure 2-3. Mean (+/SE) A)antioxidant capacity a nd B) total antioxidant ca pacity in control and fertilized Ilex vomitoria cultivar ‘Nana’ individuals obtained from the second harvest of this study (analysis based on paired samples, N=6).

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43 Figure 2-4. Relationship between % full sun and caffeine concentrati on (% dry weight of foliage) for yaupon holly ( Ilex vomitoria ) cultivar ‘Nana.’ Line indicates linear regression. Dots represent control individuals, triangl es represent fertilized individuals. caffeine conc. = -0.11 + 0.009 (% full sun) SEb = 0.005 R2 = 0.25 P = 0.1

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44 Figure 2-5. Relationship between % full sun and an tioxidant capacity, as measured by the ORAC method, for yaupon holly ( Ilex vomitoria ) cultivar ‘Nana.’ Line indicates linear regression. Dots represent control indi viduals, triangles represent fertilized individuals. ORAC = 210.4 + 16 (% full sun) SEb =3.2 R2 = 0.72 P < 0.001

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45 Figure 2-6. Mean (+1 SE) foliar caffeine c oncentrations in f our varieties of Ilex vomitoria . Lower case letters specify groupings as indicated by TukeyÂ’s Tests for multiple comparisons. b ab ab a

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46 Table 2-1. Mean ± 1 SE ORAC (oxygen radica l absorbance capacity) values for yaupon holly ( Ilex vomitoria, variety ‘Nana’ ) , wild –type yaupon ( I. vomitoria ), yerba maté ( I. paraguariensis) , and green tea ( Camellia sinensis ). N = 6 for each yaupon holly variety ‘Nana’, N = 24 for each wild-type yaupon, and N = 3 for both yerba maté and green tea. Species ORAC Value ( mol TE/g) Yaupon (‘Nana’) Unfertilized 608 ± 61.5 Yaupon (‘Nana’) N-Fertilized 670 ± 87.8 Yaupon (wild-type) Unfertilized 987 ± 83.2a Yaupon (wild-type) N-Fertilized 1034 ± 78.3 a Yerba Maté 1239 ± 55.7b Green Tea 1346 ± 60.0b a values from Chapter 1. b values from Chandra and Gonzalez de Mejia (2004).

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50 Johnson AF, Barbour MG (1990) Dunes and maritime forests In: Myers RL, Ewel JJ (eds) Ecosystems of Florida. The University of Central Florida Press, Orlando, FL, pp 429 – 480 Kaakeh W, Pfeiffer DG, Marini RP (1992) Co mbined effects of spirea aphid (Homptera Aphididae) and nitrogen fertilization on shoot growth, dry matter accumulation, and carbohydrate concentration in young a pple trees. J Econ Entomol 85:496-506 Kannan D, Paliwal K (1997) Fe rtilization response om gr owth, photosynthesis, starch accumulation. And leaf nitrogen status of Cassia siamea Lam, seedlings under nursery conditions. J Sustain For 4:141-157 Kervinen T, Peltonen S, Teeri TH, Karjal ainen R (1998) Differential expression of phenylalanine ammonia-lyase genes in barley in duced by fungal infection or elicitors. New Phytol 139:292-300 Koricheva J, Larsson S, Haukioja E, Keinanen M (1998) Regulation of woody plant secondary metabolism by resource availability: hypothesi s testing by means of meta-analysis. Oikos 83:212-226 Kumar NS, Hewavitharanage P, Adik aram NKB (1995) Attack on tea by Xyleborus fornicatus : inhibition of the symbiote, Monacrosporium ambrosium, by caffeine. Phytochemistry 4:1113-1116 Lou Y, Baldwin IT (2004) Nitrogen supply influe nces herbivore-induced direct and indirect defenses and transcriptional responses in Nicotiana attnuata . Plant Physiol 135:496 – 506 Mattson WJ (1980) Herbivory in relation to plan t nitrogen content. Annu Rev Ecol Syst 11:119161 Mazzafera P (1994) Caffeine, theobromin e, and theophylline distribution in Ilex paraguariensis . Rev Bras Fisiol Veg 6:149-151 Merrill WL (1979) The beloved tr ee. In: Hudson CM (ed) Black Drink: A Native American Tea. The University of Georgia Press, Athens, GA. pp 40-82 Montagnon C, Guyot B, Cilas C, Leroy T (1998) Genetic parameters of several biochemical compounds from green coffee, Coffea canephora . Plant Breeding 117:576-578 Nathanson, JA (1984) Caffeine and related met hylxanthines: possible naturally occurring pesticides. Science 226:184-187 Nitao JK, Zangerl AR, Berenbaum MR (2002) CNB: Requiescat in pace? Oikos 98:540-546 O’Brien TG, Kinnaird MF (2003) Caffein e and conservation. Science 300:587 O’Brien TG, Kinnaird MF (2004) Response to: Conservation policy in coffee landscapes. Science 303:625-626

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52 Stewart AJ, Mullen W, Crozier A (2005) Online high performance liquid chromatography analysis of the antioxidant activity of phe nolic compounds in green and black tea. Mol Nutr Food Res 49: 52-60 Stitt M, Krapp A (1999) The interaction between elevated carbon dioxide and nitrogen nutrition: the physiological and molecular ba ckground. Plant Cell Environ 22:583-621 Sturtevant WC (1979) Black dr ink and other caffeine-containi ng beverages among non-Indians. In: Hudson CM (ed) Black Dri nk: A Native American Tea. The University of Georgia Press, Athens, GA. pp 150-165 Sultan SE (2000) Phenotypic plasticity for plant development, function and life history. Trends Plant Sci 5:537-542 Suzuki T, Ashihara H, Waller GR (1992) Purine and purine alkaloid metabolism in Camellia and Coffea plants. Phytochem 31(2575-2584) Swain T, Hillis WE (1958) The phenolic constituents of Prunus domestica I. The quantitative analysis of phenolic constituents. J Sci Food Agr 10:63-68 Talcott ST, Howard LR, Brenes CH (2000) Antioxi dant changes and sensory properties of carrot puree processed with and without peride rm tissue. J Agri Food Chem 48:1315-1321 Terao J, Piskula M, Yao Q (1994) Protective eff ect of epicatechin, epicatechin gallate, and quercetin on lipid peroxida tion in phospholipids bilayers . Arch Biochem Biophys 308:278284 Tuomi J, Niemelä P, Chapin FS, Bryant JP, Siré n, S. (1998) Defensive responses of trees in relation to their carbon/nutrient balance. In: Mattson J, Levi eux J, Bernard-Dagan C (eds) Mechanisms of Woody Plant Defenses Agains t Insects: Search for Pattern. SpringerVerlag, NY. pp 57-72 Vandemeer J (2003) Tropical Agroec osystems. CRC Press, Boca Raton, FL van der Graff E, Hooykass P, Lein W, Lerchl J, Kunze G, Sonnewald U, Boldt R (2004) Molecular analysis of “de novo” purine bios ynthesis in solanace ous species and in Arabidopsis thaliana. Front Biosci 9:1803-1816 Weaver LM, Herrmann KM (1997) Dynamics of th e shikimate pathway in plants. Trends Plant Sci 2:1360-1385 Weinberg BA, Bealer BK (2002) The World of Caffeine: The Science and Culture of the World’s Most Popular Drug. Routledge Press, New York, NY Yokozawa T, Dong E, Nakagawa T, Kashiwagi H, Nakagawa H, Takeuchi S, Chung HY (1998) In vitro and in vivo studies on the radical-s cavenging activity of tea. J Agri Food Chem 46:2143-2150

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54 BIOGRAPHICAL SKETCH Matthew Palumbo was born in Manhasset, New York on September 20, 1970. His love of nature began when he was three years old with wa lks through the forest in rural New Jersey with his father. He distinctly remembers being jo ined on one of these walks by his mother who subsequently had to turn back because she was too pregnant (with his brother) to balance herself on a log used to cross the creek th at crossed their wooded path. Hi s love of nature was fostered during childhood when his father brought home a ga rter snake from Vermont that became the family pet, which was followed with collections of fish, newts, fresh water crabs, and more snakes. Although music became his passion duri ng high school and continues to be a great influence, he became enamored with anthropology, biochemistry, and ultimately botany during his undergraduate education, which he finish ed at SUNY at Stony Brook in 1998 with a bachelor’s degree in biochemistry. Matt then to ok a job as a chemist for a vitamin and herbal supplement company to increase his knowledge a nd skills in phytochemistry while taking classes at the New York Botanical Ga rdens and reading books on tropic al biology and ethnobotany in his spare time. After taking a year of classes as a postbaccalaureate at the Un iversity of Florida, he began a Master’s degree pr ogram in botany there in Janu ary 2004, which he finished in December 2006. During the last year of his maste r’s program, he had the opportunity to teach a course called “Plants in Human Affairs.” He found that teaching is hard but rewarding work. He plans to continue his resear ch on the ecology and ethnobotany of plant secondary metabolites while pursuing a doctoral degrees and plans to teach as the oppor tunity arises.