Plants are exposed to diverse environmental stresses, which can lead to the accumulation of harmful reactive oxygen species (ROS). To prevent cellular damage, plants have evolved diverse antioxidant compounds and mechanisms to scavenge and remove ROS. My research aimed to determine if condensed tannins (CTs) function as in vivo antioxidants in plants. CTs are abundant plant secondary metabolites and are well-known for their strong in vitro antioxidant activity, but their function as antioxidants in planta has not previously been investigated. I used transgenic hybrid poplar (Populus tremula x tremuloides) with high (MYB134- and MYB115-overexpressing) and low (MYB134-RNAi) leaf CT content. Three different abiotic stresses were used to induce oxidative stress in the plants: methyl viologen (MV), drought, or UV-B stress. Oxidative stress can damage the plant's photosystems, and this damage was assessed using chlorophyll fluorescence. I employed light-adapted (Fq’/Fm’) and dark-adapted (Fv/Fm) parameters of chlorophyll fluorescence and monitored photosystem II function during each stress. Under all three stresses, the high-CT transgenics retained greater chlorophyll fluorescence, demonstrating reduced photosystem II damage, compared to wild-type plants. Oxidative damage was measured by quantifying malondialdehyde (MDA), and hydrogen peroxide (H2O2) was quantified as a measure of ROS accumulation. High-CT plants consistently accumulated less H2O2 and MDA than wild-type plants before and after each stress. MYB134-RNAi plants showed the converse effects, as predicted by lower CT concentrations, with reduced photosystem function and increased levels of H2O2 and MDA compared to wild-type following each stress. Overall, this work demonstrates that CTs can function as in planta antioxidants and can aid in protection against oxidative damage. My work provides the first evidence for an antioxidant function of CTs in living plants exposed to stress. / Graduate / 2020-12-18

Condensed tannins or proanthocyanidins (CTs) are polymeric flavonoids and common plant secondary metabolites. They are defined by their protein binding capacity, and anti-oxidant and metal chelating properties. Known functions of CTs include anti-herbivore and anti-microbial defenses. Chemical ecology studies, especially on CTs, have focussed almost exclusively on foliar chemistry and above-ground ecological interactions. However, CT concentrations in roots can match and far exceed those found in leaves, particularly in woody plant species. This suggests that CTs are also likely to have important ecological and physiological roles below-ground. A review of the literature suggests three potential roles of root CTs: 1) defense against soil pathogens and root herbivores; 2) facilitating adaptation to toxic soils through heavy metal chelation, and; 3) facilitating or modulating nutrient uptake through the binding of nutrient cations. In this thesis the relationship between root CTs and nutrient uptake will be analysed in P. tremula x alba. Localization of CTs was determined through both quantitative and histochemical techniques. Quantitation of CTs with the 1-butanol:HCl assay clearly showed that CT concentration was higher in the white root zone compared to the brown root zone or cork zone. This is inconsistent with the term ‘condensed tannin zone’ that is commonly used to describe the macroscopic brown zone of the root between the white zone and the cork zone in Pinus. The tissue specific localization of CTs in roots was determined using the CT-specific stain, 4-dimethylaminocinnamaldehyde (4-DMACA), on embedded longitudinal and fresh cross sections. These stained sections showed that CTs were present in cells on the root surface, specifically accumulating in the root cap and epidermal cells. CTs were also sporadically present in the cortical cells of the young, white root zones. CT concentration declined as distance from the root tip increased. The pattern of CT distribution, as indicated by intensity of the staining, corresponded directly to the quantitative assay results. Growing poplars under low nitrogen availability stimulated higher root CT concentrations than when they were grown under high nitrogen availability, suggesting a link between nitrogen and CTs. Since CTs are known to bind cations in vivo, I hypothesized that root CTs may modulate or facilitate nutrient uptake by binding positively charged nutrient ions. The Microelectrode Ion Flux Measurement (MIFE) technique was used to analyse specific fluxes of NH4+, NO3- and Ca2+ at specific root locations in poplar roots, and test for spatial correlation with CT localization. This technique was also used to compare the NH4+, NO3- and Ca2+ fluxes in transgenic high-CT and wild-type lines of Populus tremula x alba to test the impact of CTs on nutrient uptake directly. No correlation was found between fluxes of NH4+, NO3- or Ca2+ and CT distribution. The data indicate that root CTs do not influence nutrient uptake in Populus. This study provides data regarding the pattern of CT distribution as well as fundamental information on nutrient uptake in P. tremula x alba roots. It provides new knowledge that will be used to stimulate investigations on other potential roles of root CTs. / Graduate

Plants containing condensed tannins (CT) represent an alternative feed resource for ruminants. However, limited information regarding nutrient disappearance from these plants is available. Two experiments were conducted to evaluate phosphorus (P) and other nutrient disappearance from plants containing CT. In the first experiment, nutrient disappearance from three native Texas species (Acacia angustissima var. hirta, Desmodium paniculatum, Smilax bona-nox, and Medicago sativa as control) were evaluated using the mobile nylon bag technique. For the second experiment, ruminal degradation parameters, ruminal and post-ruminal disappearance of P and other nutrients from a browse containing CT (Quercus virginiana) were compared to species without CT (Cynodon dactylon cv. Tifton 85, and Medicago sativa). Results from the first experiment indicate that the proportion of nutrient that disappeared during rumen, pepsin/HCl, or intestinal incubation differed among plant species and nutrient evaluated (P<0.05) and did not appear to be directly related to relative CT concentrations. Dry matter (DM), inorganic matter (IM), and organic matter (OM) disappearance were greater (P

Condensed tannins are wide-spread polyphenols with diverse ecological functions, including defense against herbivores and microbial pathogens. In poplar, condensed tannin synthesis is induced by a variety of biotic and abiotic stresses. The objective of this study was to determine the function of the R2R3 MYB transcription factor MYB115 in the regulation of condensed tannin synthesis. MYB115 was shown to be induced by wounding along with tannin biosynthetic genes and shows sequence similarity to characterized regulators of tannin synthesis in grape and persimmon suggesting that it functions in the regulation of condensed tannin synthesis. To analyze the function of MYB115, transgenic plants overexpressing MYB115 were generated and showed enhanced accumulation of condensed tannins and higher expression of flavonoid biosynthetic genes involved in condensed tannin biosynthesis compared to wild-type control plants. In promoter activation assays, MYB115 activated the promoter of a tannin-specific biosynthetic enzyme, anthocyanidin reductase. This suggests that MYB115 acts as a regulator of condensed tannin synthesis. MYB115 overexpressors showed additional changes to phenolic metabolism, including changes in levels of phenolic glycosides and hydroxycinnamic acids. These results indicate an important role of MYB115 in the regulation of the condensed tannin pathway in poplar. / Graduate / 0817 / 0307 / 0487 / frankla@uvic.ca

Fireweed (Senecio madagascariensis) is a noxious and invasive weed affecting pastures in Hawaii, Australia, and South America. Fireweed contains compounds called pyrrolizidine alkaloids that are toxic to most grazing mammals. Toxic effects to cattle include irreversible damage to liver cells, hardening of the liver, and loss of liver function, which may lead to jaundice, swelling, and the accumulation of fluids in the stomach and other physiological malfunctions. External effects include rough appearance, diarrhea, low energy and dullness, photosensitization, and abnormal behavior, many of which can lead to death. Fireweed also can reduce pasture productivity by as much as 30-40%, particularly in Hawaii. As a result of these adverse effects on cattle and pasture production, people are seeking ways to manage fireweed and cattle. Condensed tannins, which are common in many forage legumes, bind with other molecules such as protein and alkaloids. Complementarities among secondary compounds such as condensed tannins and alkaloids can allow animals to consume more of plant material they would otherwise avoid due to toxicity; however, there is very little information on whether tannins actually protect animals from the toxic effect of alkaloids like the ones found in fireweed. The effects of condensed tannins on the toxicity of fireweed to cattle were examined in two stages of a research project that included studies in the lab (in vitro) and in live animals (in vivo). The in vitro studies showed that tannins bound pyrrolizidine alkaloids in cattle rumen fluid and binding was highest when mixed with tannins at 8%, with some effect at 12% as well, by weight of fireweed. The in vivo studies were inconclusive as animals from both the Fireweed and Fireweed-Tannin group demonstrated ill-effects brought on by the fireweed. The data gathered in the form of blood tests and liver tests did not identify a clear protective effect provided by having tannin in the diet, but the results likely were affected by the way we conducted the research, which involved dosing animals daily with large amounts of plant material. The findings also illustrate how strongly cattle avoid eating fireweed while grazing on pasture. In summary, management to reduce fireweed effects on cattle should focus on improving pasture diversity and resiliency, on multi-species grazing to take advantage of the innate resistance of sheep and goats to fireweed’s toxicity, and on ways to enable cattle to utilize fireweed.

Master of Science / Department of Animal Sciences and Industry / KC Olson / Our objective was to determine the effects of supplemental corn steep liquor (CSL) on voluntary selection of sericea lespedeza (SL) by beef cows grazing native tallgrass range. Dietary botanical composition of cows (n = 145; initial BW = 579 ± 91 kg) was evaluated during a 150-d grazing period (5/1 to 10/1). Native pastures (n = 9; 50 ± 17 ha) infested by SL (average SL biomass = 37% of total forage biomass) were assigned randomly to 1 of 2 treatments: grazing by unsupplemented cows or grazing by cows supplemented with CSL (1.79 kg DM • cow-1 • d-1; 45% DM, 34% CP). Cows were assigned randomly to treatment and pasture (stocking rate = 0.5 ha/AUM). Concentration and protein-binding capacity of condensed tannins (CT) in SL were measured monthly. Fecal samples were collected from each cow on 6/1, 7/1, 8/1, 9/1, and 10/1. Herbivory of SL was estimated along line transects in October. Plant fragments in fecal samples were quantified via a microhistological technique; fragment prevalence in fecal material was assumed to equal botanical composition of the diet. Concentration and protein-binding capacity of CT in SL were greatest (P < 0.01) on 8/1 and 9/1, respectively. The proportion of individual SL plants showing evidence of herbivory tended to be greater (P = 0.09) on pastures grazed by supplemented cows compared to pastures grazed by unsupplemented cows (94 vs. 80% of SL stems, respectively). Prevalence of SL in beef cow diets was influenced (P < 0.01) by CSL supplementation and by month. Prevalence of SL in beef cow diets was not different (P ≥ 0.35) between treatments when concentration and protein-binding capacity of CT were relatively low (6/1, 7/1, and 10/1). In contrast, supplemented cows selected more (P < 0.01) SL than unsupplemented cows when concentration and protein-binding capacity of CT were greatest (8/1 and 9/1). We interpreted these data to suggest that voluntary selection of SL by beef cows was inversely related to concentration of CT; moreover, supplemental CSL stimulated voluntary selection of SL during periods of high CT concentration. Supplemental CSL did not influence selection of other plant species that were monitored.

Condensed tannins (CT) are polyphenolic compounds that have demonstrated biological activities in ruminants including suppression of enteric methane (CH4) production, protein binding and suppression of gastrointestinal nematode (GIN) infections. Some forage CT have been reported to be biologically active, whereas others have demonstrated no biological activity at all. While the chemical structure of CT has been postulated to be a key contributing factor affecting biological activity, the specific factors that determine whether or not CT from a specific forage have bioactive properties remain unknown. Results from previous studies have shown that as molecular weight of CT increases, CT biological activity also increases. Others have reported no effect of CT molecular weight on biological activity. The relationship between molecular weight of CT and CT biological activity remains inconclusive. The effect of molecular weight of CT from a variety of warm-season perennial legumes commonly consumed by ruminants on biological activity has not been adequately explored. The objectives of this study were to determine if molecular weight of CT from warm-season perennial legumes could predict the biological activity of CT relative to suppression of enteric CH4 production, protein-binding ability (PB) and anthelmintic activity, and to compare the biological activity of CT from native warm-season perennial legumes to that of the introduced species Lespedeza cuneata, a plant that has gained attention in recent years due its anthelmintic properties. All or a combination of the following warm-season perennial legume species were evaluated for in vitro gas production, protein-precipitable phenolics (PPP) and PB, and percent larval migration inhibition (LMI). Eight North American native warm-season perennial legumes: Leucaena retusa Benth. (littleleaf leadtree), Desmanthus illinoensis (Michx.) MacMill. Ex B.L. Rob. & Fernald (Illinois bundleflower), Lespedeza stuevei Nutt. (tall lespedeza), Mimosa strigillosa Torr. & A. Gray (powderpuff), Neptunia lutea (Leavenworth) Benth. (yellow puff), two ecotypes of Acacia angustissima var. hirta (Nutt.) B.L. Rob (prairie acacia), Desmodium paniculatum (L.) DC. var. paniculatum (panicledleaf ticktrefoil), and two introduced legumes: Arachis glabrata Benth. (rhizoma peanut) and Lespedeza cuneata (Dum. Cours.) G. Don (sericea lespedeza) were included. In vitro CH4 production regressed on CT MW resulted in a R2 of 0.0009 (P = 0.80). There was no correlation between PPP or PB and MW of CT (R^2 0.11; P = 0.17 and R^2 0.02; P = 0.54, respectively). There was a weak correlation between CT MW and percent LMI (R^2 0.34; P = 0.05). The results of our study strongly suggested that CT MW does not explain the biological activities of enteric methane suppression or protein-binding ability. Condensed tannin MW may be involved in anthelmintic activity of CT from the forage legumes surveyed. North American native legumes containing biologically active CT, as compared to introduced species, were identified as having promise for use in ruminant diets.

Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production.

Secondary plant metabolites have recently been gaining interest in livestock production systems following the ban of in-feed antibiotics within the European Union. The rise in antimicrobial resistance found in pathogenic and non-pathogenic bacteria has lead to increased interest in the research community regarding the use of phythochemicals as an alternative to antibiotics. The purpose of this research was to evaluate the impact of including phytochemicals in a livestock production system. Specifically, a high tannin-containing forage, sainfoin (Onobrychis viciifolia), was evaluated in vitro for its antimicrobial effect on Escherichia coli. We determined that phytochemicals alone are not as inhibitory as synthetic antibiotics. Thus, the use of combination therapy to deter the development of antimicrobial resistance was evaluated. A myriad of plant compounds were screened for their synergistic interactions with ciprofloxacin. Geraniol, an essential oil, was identified to possess good antimicrobial activity and synergistic interactions with ciprofloxacin. Therefore the effect of long term exposure to both ciprofloxacin and geraniol were examined. Results demonstrated that once an antimicrobial concentration threshold was reached, resistance to ciprofloxacin increased markedly in the presence of both geraniol and ciprofloxacin. Finally, an in vivo trial was conducted in which forty steers were fed sainfoin or alfalfa over a 9-week period to evaluate its ability to reduce E. coli shedding and its impact on gut microbiota in the context of popular theoretical ecology concepts. Results from the in vivo study indicate that sainfoin was able to promote a slight decrease in generic E. coli shedding which could be maintained throughout the trial. Using high-throughput sequencing, the effect of sainfoin on the microbial ecosystem of the ruminant gut was evaluated. Sainfoin induced a significant shift in the microbial community structure of the rumen and to a lesser extent in the hindgut. Using ecology theories, a hypothesis was formulated regarding the mechanisms that mediate the development of tolerance and the fundamental ecological processes controlling microbial population shifts. Understanding how the gut ecosystem functions and predicting its behaviour in the presence of various fluctuating environmental conditions will enable more efficient manipulation of the rumen and promote best management practices in livestock production.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol) / This study evaluated the nutritional quality of the forage plants in Namaqualand Granite Renosterveld vegetation in the Kamiesberg uplands of the Leliefontein communal area, South Africa. Determining the quality of forages is one of the most important factors necessary for the effective management of rangelands as it impacts on the nutrient needs of animals and consequently, the grazing capacity in rangelands. The edible portions of various forage plants were collected in the wet and dry seasons in 2012 and 2013 after which the nutritional quality (mineral nutrient content; crude protein (CP); fibre, neutral detergent fibre (NDF) and acid detergent fibre (ADF); dry matter digestibility (DMD); dry matter intake (DMI); metabolizeable energy (ME); and relative forage value (RFV)) as well as the anti-nutritional quality (total phenolics (TP); condensed tannins (CT); and silicon (Si) concentrations) of the plants were determined. Plants were thereafter grouped into their respective growth forms: grasses (15 species), herbs (15 species), leaf succulents (17 species), non-succulents (134 species), reeds (7 species), trees (8 species) and stem succulents (2 species) for statistical analyses. There were generally only a few significant differences for each forage growth form, when comparing the nutritional and anti-nutritional qualities between the two seasons. Certain forage types such as leaf succulents were found to have a high nutritional value in terms of their mineral nutrient content, CP, DMD and ME , but were also found to contain high concentrations of one or more anti-nutritional factors. However, within each season, results showed that for both the nutritional quality and anti-nutritional quality, there was a significant difference between the different growth forms within each season. This suggests that both of these quality parameters are essential in order to draw meaningful conclusions regarding forage quality of these semi-arid rangeland plants. Further research is needed at the species level to determine what plant species are the most nutritious in terms of both nutritional and anti-nutritional quality in order to inform the potential production of these species on a commercial scale.