Theses and Dissertations at Montana State University (MSU)
Permanent URI for this collectionhttps://scholarworks.montana.edu/handle/1/733
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Item The genetic characterization of a nil polyphenol oxidase (PPO) trait for the improvement of end product quality in wheat (Triticum aestivum)(Montana State University - Bozeman, College of Agriculture, 2014) Hystad, Steven Michael; Chairperson, Graduate Committee: Michael J. GirouxWheat (Triticum aestivum) polyphenol oxidase (PPO) contributes to the time dependent discoloration of Asian noodles. Wheat contains multiple paralogous and orthologous PPO genes expressed in wheat kernels, Ppo-A1, Ppo-D1, Ppo-A2, Ppo-D2, and Ppo-B2. To date, wheat improvement efforts have focused on breeding cultivars containing Ppo-D1 and Ppo-A1 alleles conferring reduced PPO activity. A major impediment to wheat quality improvement is a lack of additional PPO alleles conferring reduced kernel PPO. Thus, the discovery of novel low or null PPO alleles is critical to further reduce PPO and improve noodle color stability. The objective of this study was to 1) Characterize mutations present in a low PPO line 07OR1074 and create molecular markers associated with newly characterized alleles; 2) assess the allelic impact of the newly characterized alleles on kernel PPO activity and upon Chinese white salted noodle quality. To characterize additional mutations present and develop molecular markers, genomic clones were created for each known PPO gene and sequenced. Previously reported very low PPO line, 07OR1074, was found to contain a novel allele at Ppo-A2 and null alleles at Ppo-A1 and Ppo-D1 loci and molecular markers were created that encompassed each mutation. To determine the allelic impact of the new alleles on kernel PPO activity, F3:4 lines were genotyped with the novel molecular markers, grown in the field in Bozeman, Montana, and assayed for kernel PPO activity. Evaluation of lines indicated a substantial genotypic effect on PPO with Ppo-A1 and Ppo-D1 loci contributing significantly (P <.001) to total PPO. To determine the effect of the null PPO alleles upon noodle quality Chinese white salted noodles were produced from both refined white flour and whole wheat flour. The results clearly demonstrate that the null PPO alleles at Ppo-A1 and Ppo-D1 loci produce noodles that are brighter (greater L*), more red (greater a*), and more yellow (greater b*) at 24 and 48 hours. These results show that mutations in Ppo-A1 and Ppo-D1 genes are important to lowering overall wheat kernel PPO activity producing a more desirable and marketable product.Item Expression and characterization of copper-containing proteins: galactose oxidase and tyrosinase(Montana State University - Bozeman, College of Letters & Science, 2002) Kamlin, Ejan MarieItem Cloning and expression of bovine p47-phox and p67-phox(Montana State University - Bozeman, College of Agriculture, 1999) Bunger, Peggy Lee OhmstedeItem Molecular and biochemical characterization of wheat (Triticum aestivum. L) polyphenol oxidases (PPOs)(Montana State University - Bozeman, College of Agriculture, 2005) Jukanti, Aravind Kumar; Chairperson, Graduate Committee: Andreas Fischer.Polyphenol oxidases (PPOs) from several plant species, including wheat, have been implicated in the undesirable brown discoloration of food products. Wheat (Triticum aestivum L.) represents an interesting system to advance our understanding of plant PPO function for two important reasons, namely (1) the large size an complexity of its (allohexaploid) genome, and (2) its economic importance. Prior to this study, the molecular and biochemical properties of wheat PPOs were largely unknown. To remedy this situation, we have performed several BLAST searches of expressed sequence tag (EST) databases, using a known wheat PPO sequence as a search tool. This study suggested the presence of at least six PPO genes in hexaploid wheat, falling into two different phylogenetic clusters of three genes each. Presence of a wheat PPO multigene family was confirmed by Southern blotting. A combination of biochemical (enzyme purification and mass spectrometric analysis) and molecular (Northern) approaches confirmed that members of one cluster are not expressed in the developing kernels and senescing flag leaves, while regulation of one or several members of the other gene cluster controls PPO activity in these tissues. Our data, including immunoblotting and enzyme activity studies, further indicated that wheat PPOs are synthesized as inactive precursor (early kernel development) which are proteolytically processed and activated as the kernels mature. Activation of PPO precursor proteins was also demonstrated in vitro, in presence of purified trypsin. In these experiments, PPO activity increased during the first four hours and remained stable thereafter, indicating that the protein domains responsible for catalytic activity are quite stable. Research performed as a part of this dissertation has also demonstrated that wheat PPO activity is influenced by strong anionic detergents such as SDS and N-lauroylsarcosine. The corresponding experiments indicated that these detergents influenced both enzyme extraction and activity, at least in high-PPO wheat varieties. This work also has practical aspects, making PPO assays (as used by breeders for germplasm screening) more robust. In conclusion, as a result of this dissertation, wheat PPOs have emerged as a fascinating example of a plant multigene family with complex transcriptional and posttranscriptional regulation.