Browsing by Author "Wu, Hao, 1987-"
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Item The role of general control non-derepressible 2 in maize growth, development and stress response.(2014-09-05) Jia, Mo, 1984-; Gibbon, Bryan C.; Baylor University. Dept. of Biology.; Larkins, Brian A.; Jung, Rudolf.; Clay, Kasi L.; Wu, Hao, 1987-; Smith, Sonya S.; Finlayson, Scott A.; Baylor University. Dept. of Biology.Maize is among the most important food sources in the world. However, maize protein is not nutritionally balanced, limiting its value. As a result of climate change, frequent heat and drought stresses negatively affect maize yield. Therefore, it is critical to improve maize nutritional value, yield and performance under stress conditions to sustain global food security. General control non-derepressible-2 (GCN2) plays an important role in cellular responses to amino acid starvation. During amino acid starvation, GCN2 phosphorylates the α subunit of eukaryotic translation initiation factor-2 (eIF2), which enhances the translation of the transcription factor GCN4 by overcoming the inhibitory effect of upstream open reading frames (uORFs). This results in increased expression of many amino acid synthesis genes, and is known as general amino acid control. In this study, we first investigated the pleiotropic effects of maize opaque2 (o2) mutation. The o2 mutant was discovered to have enhanced essential amino acid lysine content, demonstrating nutritional superiority. We found elevated protein accumulations that could partially explain the high lysine content and altered gene expression associated with the increased insect and fungal susceptibility and the brittle endosperm texture of o2. Next, we showed that maize GCN2 kinase phosphorylated eIF2α in response to amino acid starvation in maize endosperm. It was associated with an increase of O2 protein accumulation but no alteration of O2 transcript was detected, indicating that the regulation of O2 was translational and that O2 could be a maize GCN4 ortholog. We then tested the role of GCN2 in other stress situations, such as drought. The gcn2 mutant showed increased tolerance to drought compared to wild type and had a higher level of steady state abscisic acid (ABA). RNA-Seq analysis indicated candidate genes responsible for the increased tolerance but further analysis was necessary for candidate genes involved in elevated ABA abundance. Finally, we screened for proteins involved in the localization of the 27 kDa γ- zein, which is important for the protein body formation in maize endosperm, and found that maize eIF5A was associated with γ-zein mRNA and an actin rich cytoskeleton, indicating the possible role of eIF5A in localizing γ-zein.Item The role of pullulanase and SSIII in the formation of the vitreous endosperm phenotype in Quality Protein Maize.(2015-07-20) Wu, Hao, 1987-; Gibbon, Bryan C.; Kearney, Christopher Michel, 1958-Despite being a major food source, maize does not provide a balanced source of protein, because maize lacks some essential amino acids, such as lysine and tryptophan. opaque-2 (o2) mutants highly increased the lysine content, but they were not suitable for commercial development, due to their soft and chalky endosperm. Modifier genes were introgressed into o2 maize that restored hard and vitreous endosperms, while maintaining the high lysine content and these lines were called Quality Protein Maize (QPM). In this study two candidate factors associated with the formation of the vitreous endosperm phenotype in QPM were characterized; Zpu1, encoding pullulanase-type starch debranching enzyme and SSIII, encoding Starch Synthase III. The data showed that the QPM inbred line, K0326Y, had higher pullulanase activity and SSIII abundance than W64Ao2. Recombinant inbred lines (RILs) generated from a W64Ao2 X K0326Y cross demonstrated that kernel vitreousness was positively correlated with pullulanase activity, indicating pullulanase may be one of the key factors affecting vitreousness. Pullulanase activity was highly dependent on Zpu1 alleles and SSIII alleles, indicating that SSIII might be an indirect factor associated with kernel vitreousness that modulates pullulanase activity. Structural analysis of starch isolated from endosperms of RILs showed that the starch fine structure could be altered by changes of pullulanase activity and SSIII abundance. Sequence analysis showed a single amino acid change between the Zpu1 genes derived from W64Ao2 and K0326Y. Therefore, they were cloned and recombinant proteins were used to assay enzyme activity. Pullulanase from W64Ao2 had higher activity than the K0326Y counterpart, which contradicted the native pullulanase activity comparison between W64Ao2 and K0326Y in crude endosperm extracts. Therefore, the null mutants Zpu1-204 and du1-M4 (null mutant of SSIII) were analyzed to identify possible physical or functional interaction between the two enzymes. This showed that pullulanase activity was significantly reduced in du1-M4, which also affected the thermal properties and surface characteristics of starch granules. Thus SSIII may affect the activity of other enzymes in starch biosynthetic pathway, such as pullulanase. These experiments demonstrated that pullulanase and SSIII play a role in the formation of vitreous endosperm in QPM.