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

A series of in vitro investigations was conducted to study the effects that polyphenolic compounds exerted toward rumen cellulolytic micro-organisms. The effect of the complexing of tannins with proteins on the ruminal degradation and intestinal digestibility of protein was also tested. Five polyphenolic compounds of known molecular mass were selected as representative of hydrolysable tannin (tannic acid) or their molecular components (gallic acid and ellagic acid) and components of condensed tannin (catechin and epicatechin). The effects of polyphenolic compounds on the degradation of microcrystalline cellulose (Avicel) and ryegrass and on the formation of gas and VFAs by mixed rumen micro-organisms undergoing rumen-like fermentation (consecutive batch culture, CBC) were studied. On a molar and weight basis, tannic acid was the most inhibitory compound towards cellulose degradation and on the production of VFAs followed by ellagic acid, catechin and epicatechin. Low concentrations (up to 10 mM) of gallic acid caused a significant (P < 0.05) stimulation of cellulolysis, gas and VFA formation. This was probably due to the presence of gallate-degrading bacteria in the rumen. The ability of the ruminal fungus Neocallimastix frontalis to degrade cellulose in two different media, to adhere to cellulose, to utilize glucose and to form colonies from zoospores was assessed, in the presence and absence of polyphenolic compounds. The compounds tested were more inhibitory towards fungi grown in a defined medium rather than in a peptide and rumen fluid-containing medium, presumably due to the interactions between the proteins in the medium and the tested compounds. The adhesion of three fractions of the fungi to cellulose was inhibited by the presence of phenolic compounds in the medium.

Doctor of Philosophy / Department of Animal Sciences and Industry / K.C. Olson / Sericea lespedeza (SL; Lespedeza cuneata) is classified as an invasive plant throughout the Great Plains. It infests over 600,000 acres in Kansas. Increasing grazing pressure on SL may reduce seed production and slow the spread of the plant; however, intake of SL by grazing beef cattle is poor, due to the presence of tannins in the plant. Condensed tannins reduce protein digestion by ruminants and may also decrease plant palatability. Detailed study of the appetite-suppressing effects of SL under controlled conditions is essential in order to develop appropriate strategies to increase grazing pressure on this plant. Such information could lead to a degree of biological control of this noxious weed using domestic herbivores. We compared intakes of tallgrass prairie hay by beef cows when hay was either uncontaminated or heavily contaminated by SL. Beef cows fed contaminated hay exhibited a profound aversion to compared to similar uncontaminated hay. Furthermore, differences in voluntary DMI between contaminated and uncontaminated hays of similar chemical composition were manifested rapidly after introduction of contaminated hay into beef cow diets. Supplementation with corn steep liquor (CSL) increased tolerance of beef cows for SL. It ameliorated the negative consequences of tannin consumption in a dose-dependent manner when fed to beef cows in confinement. The beef cows in our study had only limited opportunity to selectively avoid SL because it was offered in chopped form and in a mixture with other forage species. It was unknown if beef cattle supplemented with CSL would readily consume forage contaminated by SL when uncontaminated forage was available simultaneously. Therefore, we examined the effects of CSL fed to beef cows on voluntary selection of tallgrass prairie hay contaminated by SL when uncontaminated tallgrass prairie hay was also available. Supplemental CSL (0.6 kg DM/d) increased both acceptance of and tolerance for SL by beef cows. It ameliorated some of the negative consequences of tannin consumption on digestible DM intake. In addition, voluntary consumption of SL-contaminated forage increased by 25% in supplemented vs. unsupplemented beef cows. It is unknown if supplemental CSL can promote voluntary selection of actively-growing SL by beef cattle grazing native rangeland in the Kansas Flint Hills.

No description available.

To evaluate the anticancer potential of sorghum phenolic compounds, different experiments including in vitro and in vivo tests were performed. A set of 25 sorghum samples was evaluated for phenolic (total phenols, flavonoids, anthocyanins and tannins) content, hydrophilic and lipophilic antioxidant capacity using de Oxygen Radical Absorbance Capacity assay (ORAC), and screened for citotoxic properties in mammary, colon and hepatic mammalian cancer cell lines in vitro. Results indicated that there was a wide variability in the phytochemical profile among the different sorghums. Among the 25 samples, sumac sorghum bran had the highest amount of phenolic compounds, flavonoids, tannins and the highest ORAC values. It exerted the highest percent inhibition (near 100%) in mammary, colon and liver cancer cell lines. Sumac sorghum bran was selected for further investigation. Methanolic extracts from sumac whole grain, bran and tannin removed bran were tested in vitro at different concentrations in hormone dependent MCF-7 mammary cancer cells and non hormone dependent Caco2 and HepG2 colon and liver cancer cells. Results indicated that the methanolic extract from sumac bran inhibited 100% of MCF-7 cancer cells at a concentration of 0.5 mg/ml and that the citotoxic effect could be partially due to the tannin content of the extract. Concentrations of 0.5 and 1.5 mg/ml were selected for an in vivo preventive cancer study with 7,12-dymethylbenz(a)-anthracene (DMBA) induced female rats. Bran at low and high concentrations and the correspondent amount of methanol extracts were included in the diet. It was observed that sumac methanol extract at low concentration promoted tumor appearance and development, whereas sumac bran had a preventive effect, however, there were no significant differences in rats treated and un-treated with sumac. Differences between in vitro and in vivo results could be due to the degree of absorption of tannins during the in vivo experiment. To obtain additional data about the effect of sumac extracts on cancer development, a quinone reductase enzyme bioassay was performed. Methanol and hexane extracts from sumac bran induced phase II enzymes in vitro. Phytochemicals of sumac bran sorghum including phenolic compounds and lipid like compounds appeared to have potential for cancer prevention.

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

Traditional processing technologies can negatively affect polyphenolics of blueberry products. Optimization of hydrothermodynamic (HTD) processing has been used to minimize blueberry-based product quality degradation. Preliminary experiments have shown that the quality of HTD blueberry purée in terms of anthocyanin concentration and tannin content was significantly higher than that of most commercial blueberry products. HTD technology provided pasteurization with significantly lower losses of anthocyanins as compared with conventional heating. Screening of the factors has shown that holding time, temperature of pasteurization and heating rate were significant, while cavitation mode was non-significant. Response surface methodology was used to determine an optimal combination of temperature and time (92 to 95°C with holding time 0.5 to 2.0 min). Non-linear regression analysis of the shelf-life data has provided fitted models which could be used to predict changes in anthocyanin concentration and tannins for different storage periods at 4°C and at room temperature (20 to 25°C).

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

Thesis (M.S.)--Virginia Polytechnic Institute and State University, 1993. / Vita. Abstract. Includes bibliographical references (leaves 76-82). Also available via the Internet.