No description available.

No description available.

Doctor of Philosophy / Food Science Institute / Weiqun Wang / Anthocyanins are the most abundant water-soluble flavonoid pigments that are biosynthesized via the phenylpropanoid pathway in plants. Consumption of anthocyanin-rich vegetables and fruits has been linked with multiple health benefits in chronic disease prevention. This dissertation consisted of three studies as follows focused on the profiles and contents of anthocyanins in various sorghum accessions and sweetpotato leaves. Study 1: Sorghum is a rich source of various phytochemicals, but the contents of anthocyanins in various sorghum accessions are not clear. This study was to identify and quantify the anthocyanins by HPLC-DAD in selected 25 sorghum accessions with various phenotypic pericarp pigments. The predominant anthocyanins found in sorghums were 3-deoxyanthocyanidins including the unique leuteolinidin and apigeninidin analogs. The high levels of total anthocyanins were found in the red pericarp PI297139 and the brown pericarp PI221723, followed by the brown pericarp PI35038 and the yellow pericarp PI229838. There were moderate to low levels of anthocyanins observed in all the other accessions except for the white pericarp that generally contained least to undetectable amount. Although anthocyanins appeared to be associated with the pericarp color in the sorghum accessions with the highest contents in each pericarp pigment category, a distinguishable diversity of anthocyanin contents was presented among and between the phenotypic pericarp colors, suggesting other colorful phytochemicals such as carotenoids might be contributed. Establishing a database of anthocyanin profile and diversity in sorghum accessions with various pericarp pigments may lead to the development of novel functional sorghum products with active anthocyanin-enriched health benefits. Study 2: As phytochemical-enriched edible greens, sweetpotato (Ipomoea batatas L.) leaves have become popular. However, the profile and content of phytochemicals in sweetpotato leaves are mostly unknown. We previously bred a purple-fleshed sweetpotato P40 that demonstrated cancer prevention due to high levels of anthocyanins in the tuberous roots. The objectives of this study were to identify and quantify anthocyanins in P40 leaves when compared with the white-fleshed Bonita and orange-fleshed Beauregard. The mature leaves of P40 at 6-week vine stage were collected and extracted for anthocyanin analysis by HPLC-MS/MS. Fourteen anthocyanins, including a novel anthocyanin (peonidin 3-caffeoyl-p-coumaryl sophoroside-5-glucoside), were identified and quantitated. The contents of anthocyanins in P40 leaves (38 ± 2.9 mg/kg DW) were much lower than that in the tubers (13,100 ± 70 mg/kg DW). Furthermore, anthocyanin contents in P40 leaves were even lesser than those of the white-fleshed Bonita (448 ± 50.4 mg/kg DW) and orange-fleshed Beauregard (240 ± 60.9 mg/kg DW). Total phenolic contents as measured by Folin-Ciocalteu were 36.8 ± 4.8 mg GAE/g DW in the leaves of P40, but 46.7 ± 2.1 mg GAE/g DW in Bonita and 41.2 ± 5.0 mg GAE/g DW in Beauregard. No anthocyanin was detectable in the stems of these three sweetpotato varieties. Taken together, this study reports for the first time the profile and content of anthocyanins in the leaves of three sweetpotato varieties with a new anthocyanin identified. The unexpected lower levels of anthocyanins in the purple-fleshed sweetpotato leaves when compared with the tuberous roots advanced our existing database and also validated a diverse phenotype of anthocyanin biosynthesis between sweetpotato leaves and tubers. Study 3: As phytochemical-enriched edible greens, sweetpotato (Ipomoea batatas L.) leaves have potential health benefits. However, how anthocyanin content in sweetpotato leaves responds to harvest stages and growth conditions remains mostly unknown. In this study, we investigated the effect of harvest timing on the accumulation of anthocyanin in the leaves of several sweetpotato varieties: white-skinned and white-fleshed Bonita, red-skinned and orange-fleshed Beauregard, red-skinned and white-fleshed Murasaki, and purple-skinned and purple-fleshed P40. Anthocyanin content increased continuously in Bonita from 1st slip stage to vine stage, but P40 did not have the same response. Beauregard had most anthocyanin (592.5 ± 86.4 mg /kg DW) and total phenolic content (52.2 ± 3 mg GAE/g DW) of mature leaves at vine stage. The P40 variety had low anthocyanin and total phenolic content in the leaves although P40 tubers have the highest among these varieties. In the high tunnel studies, no significant differences in anthocyanin content were found in Beauregard leaves grown in the high tunnels versus the open field. Our study showed for the first time that anthocyanin levels were significantly affected by the growth stages. Our overall results indicate that growth stage and/or environmental factors among sweetpotato varieties affected anthocyanin content, which is highly variable and genotype-dependent. In conclusion, the three studies conducted in this dissertation provided a fundamental understanding of anthocyanin profiles and contents in various sorghum accessions with various phenotypic pericarp pigments and sweetpotato leaves in various growth stages and conditions. These results can be useful not only for the breeders but also consumers in the selection of sorghum accessions and sweetpotato varieties for anthocyanin-contained health benefits.

The genus Antirrhinum (commonly known as snapdragons) contains more than twentyfive recognised species. The genus has been divided into three morphological subsections: Antirrhinum, Streptosepalum and Kickxiella (Rothmaler, 1956). One of the major characteristics distinguishing the three subsections is flower colour. Most species in subsection Antirrhinum have dark pink or yellow flowers, Kickxiella species are white or pale pink and Streptosepalum species have yellow or pale pink flowers. All Antirrhinum species can be crossed to produce fertile hybrids which allow the genes that underlie their differences to be identified. I used quantitative trait locus (QTL) analysis on hybrids of A. majus (dark magenta flowers) and A. charidemi (pale-pink flowers) to map genomic regions underlying differences in flower colour. This identified two major-effect loci, in Linkage Group 3 (LG3) and LG7, that explained most of the differences between these species. I used near-isogenic lines (NILs) to further test involvement of two candidate genes - Rosea (Ros) in LG3, which encodes a regulator of the anthocyanin biosynthesis pathway (ABP) and Incolorata (Inc) in LG7 which encodes a rate-limiting enzyme of the ABP. In both cases, the A. majus allele increased pigmentation. Sequence differences between Ros alleles of A. majus, A. charidemi and A. molle (a Kickxiella species with white flowers) suggest that A. molle carries a ros loss-of-function mutation and that a transposon insertion in the ROS promoter might contribute to differences in expression between A. majus and A. charidemi. Ros genotypes were found to be strongly correlated with pigmentation in the corolla tube in A. majus x A. charidemi hybrids, and to a lesser extent with corolla lobe pigmentation, although NILs suggested that ROS did not correspond to the major-effect QTL indentified in LG3. I also mapped a minor-effect QTL for tube pigmentation to a region of LG4 containing the ABP structural gene Candica. Analysis of NILs revealed that Inc was not the second major-effect QTL mapped to LG7, although sequence differences were detected between Inc alleles of A. majus and A. charidemi. I was further able to narrow down the region containing the second LG7 major-effect QTL to an interval of 11 cM, between two molecular markers, which could be used to determine the likely QTL genotypes of segregating NILs. Surprisingly, several ABP genes, particularly Nivea, Inc and Pallida, were expressed at higher levels in pale flowers that were homozygous for the A. chardemi QTL allele than in their dark flowered siblings that carried an A. majus allele. This suggests that ABP genes might be up-regulated in pale flowers as part of a negative feedback mechanism. Two potential roles of the LG7 QTL are considered 1) its requirement for anthocyanin modification or transport to the vacuole, so that a build-up of cytosolic anthocyanins or their break-down products in pale flowers increases structural gene expression but cannot compensate for the overall reduction in anthocyanin, or 2) a role in promoting production of flavonols at the expense of anthocyanins.

No description available.

Proanthocyanidins (PAs), also known as condensed tannins (CTs), are oligomers or polymers of flavan-3-ols. They have a very important role in plant-environment interactions, such as defense against herbivory and pathogens. They may also be important for light stress tolerance. In poplar, PAs can make up as much as 30% of the leaf dry weight. The synthesis of PAs in poplar was demonstrated to be inducible by both abiotic and biotic stresses. The B-ring hydroxylation pattern of flavan-3-ols directly affects the structure of PAs, and many studies have shown that B-ring hydroxylation of PAs is associated with their biological functions, including effects on leaf litter decomposition rate and anti-herbivore activity. Anthocyanins are very important colour pigments in plants, and share the intermediate leucoanthocyanidin with PAs. The role of anthocyanins in plant pollination, light stress tolerance, and seed dispersal has been well studied. A change in B-ring hydroxylation pattern can modify the colour of anthocyanins dramatically and also change their biological function. Flavonoid 3’-hydroxylase and flavonoid 3’, 5’-hydroxylase (F3’H and F3’5’H) are the two enzymes involved in determining the B-ring hydroxylation pattern of both PAs and anthocyanins. The objective of this study is to characterize the possible role of flavonoid 3’, 5’-hydroxylase in PA and anthocyanin biosynthesis in poplar. A candidate F3’5’H was identified in the Populus trichocarpa genome database based on previous expression profile experiments, and called PtF3’5’H1. The predicted protein shares high sequence similarity with previously characterized F3’5’H proteins from other plants. To test the function of PtF3’5’H1 directly, transgenic hybrid poplar plants overexpressing PtF3’5’H1 were generated. Preliminary LC-MS analysis showed that the hydroxylation pattern of the PA in the transgenic poplars was not significantly modified. Likewise, overexpression of PtF3’5’H1 in poplar did not change the overall amount of PAs. These results suggest that overexpression of PtF3’5’H1 in poplar is not sufficient to modify the B-ring hydroxylation pattern of PA, and that additional factors may be required. By contrast, the transgenic PtF3’5’H1 overexpressing poplar did show an alteration in anthocyanin profile. In leaves of transgenic poplars, several putative delphinidin derivatives were observed at greater levels than in the wild type, indicating that PtF3’5’H1 participates in the anthocyanin production in poplar. However, transiently introducing PtF3’5’H1 into Nicotiana benthamiana had no effect on the anthocyanin profile in this plant. I conclude that PtF3’5’H1 is very likely to be involved in the anthocyanin synthesis in poplar, while the function of PtF3’5’H1 in poplar PA synthesis has yet to be demonstrated. / Graduate

Master of Science / Department of Human Nutrition / Weiqun Wang / Anthocyanins may prevent chronic diseases such as cancer and cardiovascular disease, however, the anthocyanin metabolites are not well elucidated. We previously selected a purple-fleshed sweet potato clone P40 that contained anthocyanins at up to 7.5 g/kg dry matter, most of which are cyanidin and peonidin derivatives. The main objective of this study is to identify anthocyanin metabolites in the mice fed 20-30% of purple sweet potato P40 (287 mg and 430 mg peonidin-3-glucoside equivalent /kg body weight) diet for 6 weeks. Plasma, liver, and feces were analyzed for anthocyanin metabolites using HPLC/MS and MALDI-TOF-MS. Fifteen hours after consumption of P40 diet, we identified 4 anthocyanin metabolites cyanidin 3,5- diglucoside; cyanidin 3-sophoroside-5-glucoside; cyanidin3-p-hydroxybenzoylsophroside-5-glucoside; and peonidin 3-p-hydroxybenzoylsophroside-5-glucoside in fecal samples. No anthocyanin metabolites were detected in plasma or liver extracts by HPLC/MS or MALDI-TOF-MS. The results indicate that anthocyanin metabolites in fecal samples might provide health benefits for colonic mucosal cells. However, the lack metabolites in both plasma and liver samples suggest a continuous intake of the anthocyanins may be required for systemic benefits due to their quick degradation and low bioavailability.

Isolation and purification of anthocyanins from black bean canning wastewater by column chromatography with macroporous resins were investigated in this study. Different adsorption materials and adsorption conditions were compared and the most effective material and adsorption conditions were selected to purify anthocyanins. Purified anthocyanins then were identified by high performance liquid chromatography electrospray tandem mass spectrometry. The most effective macroporous resin was selected by comparing the adsorption performance of five different types of macroporous resins (Diaion Hp20, Sepabeads Sp70, Sepabeads Sp207, Sepabeads Sp700, and Sepabeads Sp710). Equilibrium adsorption isotherms of five resins with wastewater were measured and analyzed using Langmuir and Freundlich isotherm models. Both Langmuir and Freundlich models could describe the adsorption process. The adsorption and desorption behaviors of anthocyanins were studied using a dynamic method on the five types of resins, and Sp700 presented the highest adsorption capacity as well as desorption capacity, indicating that Sp700 is a good candidate for purification of anthocyanins from black bean canning wastewater. The most effective adsorption conditions were tested using Sp700. Dynamic adsorption and desorption were performed in glass columns packed with Sepabead Sp700 to optimize the purification process. Temperature during adsorption and desorption (25°C and 35°C) did not significantly affect the adsorption and desorption ratio. Adsorption ratio was significantly reduced when the flow rate increased from 1.5 mL/min to 2.5 mL/min. However, desorption ratio was not affected by flow rate (from 1.5mL/min to 0.3mL/min). Ethanol concentration (from 30% to 60%) did not affect desorption ratio. Four kinds of anthocyanins were identified in black bean canning wastewater. The major anthocyanins were delphinidin 3-glucoside, petunidin 3-glucoside, and maldvidin 3-glucoside, with a small amount of petunidin 3, 5-diglucoside also in the final product.

Flavonoids are important and ubiquitous secondary metabolites and are known to participate in various developmental and stress response processes in plants. Common flavonoids include anthocyanins, proanthocyanidins and flavonols. This thesis aims to determine, at the molecular level, how the biosynthesis of flavonoids, in particular the proanthocyanidins, is regulated in poplar. Poplars accumulate large amount of flavonoids and the major flavonoid biosynthetic genes in poplar have been identified. Flavonoid biosynthesis is known to be regulated by MYB transcription factors. Previous work had identified MYB134 as a key regulator of proanthocyanidin synthesis in poplar. Here I describe experiments on five additional genes encoding MYB activators (MYB115 and MYB117), MYB repressors (MYB165 and MYB194), and one bHLH cofactor (bHLH131) as possible flavonoid regulators in poplar. The objective of this work is to determine the in planta functions of these new flavonoid regulators using reverse genetic methods, phytochemical and transcriptome analysis, to identify their target genes and to determine how these transcriptional regulators interact using promoter transactivation and yeast two- hybrid assays. MYB115 was identified as a second proanthocyanidin regulator. Similar to the effects of MYB134, overexpression of MYB115 in poplar led to increased proanthocyanidin content and upregulated flavonoid biosynthesis genes, but reduced the accumulation of salicinoids. Overexpression of repressor type MYBs, MYB165 or MYB194 led to reduced anthocyanin, salicinoid and hydroxycinnamic ester accumulation in leaves, while reducing proanthocyanidin content in roots. Transcriptome analysis demonstrated the downregulation of most flavonoid genes in these transgenics, as well as some shikimate pathway genes, confirming the broad repression function on the phenylpropanoid pathway. By contrast, MYB117 encodes an anthocyanin activator, and was shown to be specific to this branch of the flavonoid pathway. Overexpression of MYB117 in transgenic poplar increased accumulation of anthocyanin in all tissues, resulting in red poplar plants. One bHLH cofactor, bHLH131 was shown to interact with both MYB activators and repressors and required by MYB activators to activate flavonoid gene promoters. This indicate an important role of bHLH131 in the flavonoid biosynthesis. Proanthocyanidin MYB activators, MYB134 and MYB115 could activate each other. This indicates a positive feedback loop of proanthocyanidin MYB activators. Interestingly, repressor MYB165 suppressed expression of other flavonoid MYB repressors including MYB194 and MYB182, which shows a negative feedback loop of MYB repressors. The expression of bHLH131 was also regulated by MYB activators and repressors. These results reveal the complex interaction between these regulators. iii iv Unexpectedly, overexpression MYB134, MYB115 or MYB117 poplars upregulated flavonoid 3’5’-hydroxylase and cytochrome b5 genes, and lead to enhanced flavonoid B- ring hydroxylation and an increased proportion of delphinidin, myricetin Overexpression of flavonoid 3’5’-hydroxylase in poplar confirmed its function in enhancing B-ring hydroxylation. However, overexpression of cytochrome b5 in flavonoid 3’5’-hydroxylase- overexpressing plants did not further increase flavonoid B-ring hydroxylation. Thus its role in flavonoid B-ring hydroxylation remained unclear. These results show that flavonoid MYBs could also alter flavonoid structure. Together, these studies outline the complex regulatory network formed by flavonoid MYB activators and repressors, and bHLH cofactors controlling both flavonoid accumulation and structure. / Graduate / 2020-12-04

Isolation and purification of anthocyanins from black bean canning wastewater by column chromatography with macroporous resins were investigated in this study. Different adsorption materials and adsorption conditions were compared and the most effective material and adsorption conditions were selected to purify anthocyanins. Purified anthocyanins then were identified by high performance liquid chromatography electrospray tandem mass spectrometry. The most effective macroporous resin was selected by comparing the adsorption performance of five different types of macroporous resins (Diaion Hp20, Sepabeads Sp70, Sepabeads Sp207, Sepabeads Sp700, and Sepabeads Sp710). Equilibrium adsorption isotherms of five resins with wastewater were measured and analyzed using Langmuir and Freundlich isotherm models. Both Langmuir and Freundlich models could describe the adsorption process. The adsorption and desorption behaviors of anthocyanins were studied using a dynamic method on the five types of resins, and Sp700 presented the highest adsorption capacity as well as desorption capacity, indicating that Sp700 is a good candidate for purification of anthocyanins from black bean canning wastewater. The most effective adsorption conditions were tested using Sp700. Dynamic adsorption and desorption were performed in glass columns packed with Sepabead Sp700 to optimize the purification process. Temperature during adsorption and desorption (25°C and 35°C) did not significantly affect the adsorption and desorption ratio. Adsorption ratio was significantly reduced when the flow rate increased from 1.5 mL/min to 2.5 mL/min. However, desorption ratio was not affected by flow rate (from 1.5mL/min to 0.3mL/min). Ethanol concentration (from 30% to 60%) did not affect desorption ratio. Four kinds of anthocyanins were identified in black bean canning wastewater. The major anthocyanins were delphinidin 3-glucoside, petunidin 3-glucoside, and maldvidin 3-glucoside, with a small amount of petunidin 3, 5-diglucoside also in the final product.