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theses

This dissertation is divided into two chapters with a common theme of investigating the role of several genes in seed development. Studies of the genetic basis of corn seed development provide not only answers to basic biological questions, but also have significant implications for nutritional and industrial uses. For example, the relative concentrations of different types of storage proteins in maize, called zeins, greatly affect the amount of essential amino acids lysine, methionine, and tryptophan, which are important for human and animal nutrition (Mertz, et al. 1964; Messing and Fisher 1991). The interaction of protein bodies with starch granules also affects kernel hardness and consequently the transportation and storage of corn grains (Wu, et al. 2010). The quantity and quality of another important household commodity – corn oil – also depends on the biosynthesis of triacylglycerols and their storage into lipid bodies which are mainly found in the embryo (White and Weber 2003). Clearly, understanding the many pathways involved in seed development is an important step towards improving its uses. In the first chapter, the conservation of regulatory factors controlling gene expression of zeins throughout the Poaceae were investigated by taking advantage of oat-maize addition lines or OMAs. Oats and maize belong to two different subfamilies of the Poaceae, but it was possible to cross pollinate the two and obtain seeds, where one maize chromosome at a time can be added to the whole set of oat chromosomes (Kynast, et al. 2001; Rines, et al. 2009). Therefore, one can examine whether oat has regulatory factors that can cause the expression of genes added by the single maize chromosomes, but regulated by a different maize chromosome. The results showed that recently diverged genes of the prolamin gene family, the α-, β-, and δ-zeins, were not expressed, whereas the older γ-zein genes were expressed. Further studies also showed that the oat homolog of a known regulator of zein gene expression called Prolamin box binding factor 1 (Pbf1) was able to trans-activate γ-zein expression in transient expression assays, indicating that it can substitute for the function of its maize homolog. The wheat Dx5 gene, presumably the founding member of the prolamin gene family (Xu and Messing 2009), is also expressed in a maize transgenic line, even when zein transcription factors, Pbf1 and O2, are knocked down. Overall, our data indicates that the regulation of gene expression of old copies of seed protein genes is conserved, whereas the regulation of younger copies seems to have diverged. Mutant collections are also great resources for identifying genes involved in seed development. Many researchers have used two mutant resources to study maize seed development – a collection of defective kernel (dek) mutants developed from EMS mutagenesis and mutants from Mutator insertion collections. Some of the genes that were identified using these two resources encode a heat shock binding protein (Fu, et al. 2002), a chloroplast DNA polymerase (Udy, et al. 2012), an RNA splicing factor (Fouquet, et al. 2011), and several enzymes (Lid, et al. 2002; Wang, et al. 2014). The second chapter uses a new insertion mutant resource that has been developed based on a Ds transposable element tagged with GFP (Dsg) (Li, et al. 2013). Selection of a seed with a defective kernel phenotype has led to the identification of dek38-Dsg, a recessive lethal mutant that encodes a co-chaperone protein called Tel2-interacting protein 2 (TTI2). TTI2 interacts with two other co-chaperones called Telomere maintenance 2 (Tel2) and Tel2-interacting protein 1 (TTI1) to form the TTT complex that is required to maintain steady-state levels of phosphatidylinositol 3-kinase-related kinases (PIKKs) which are essential for development (Hurov, et al. 2010; Takai, et al. 2010). As a co-chaperone for HSP90, the TTT complex can interact directly with PIKKs to aid in their proper protein folding and assembly into functional complexes. Reversion analysis and multiple Dsg excision footprint alleles established the linkage of the gene to the phenotype. Histological sections of developing seeds show that many aspects of development are affected in dek38-Dsg. The arrest of embryo development at the transition stage is similar to the PIKK Target of rapamycin (TOR) mutation in Arabidopsis and is consistent with reduced level of TOR protein in dek38-Dsg. Pollen transmission problems, as shown by the significantly lower number of GFP kernels when dek38-Dsg is used as a male, indicate that TTI2 is important for male reproductive cell development. Cloning of maize Tel2 and Tti1 homologs and yeast two-hybrid assays show that the interaction of TEL2 to TTI1 and TTI2 is conserved in maize. Overall, the results open up new lines of investigations into the roles of co-chaperones in seed development, and show the advantages of the Dsg insertion collection in maize for functional analysis of a gene. To sum up, my dissertation presented evidence of the conservation of gene expression in older copies of seed storage protein genes in maize, and showed the utility of a new mutant resource in maize by characterizing the effects of Tti2 mutation for the first time in plants. Our findings provide new avenues to the roles of gene expression and genome evolution, as well as the role of co-chaperones in seed development. For example, how do promoters or DNA binding proteins diverge after gene amplification? What are the direct targets of Tti2 during seed development? The approaches taken here have advanced our understanding of the genetic basis of seed development and allowed us to suggest further directions in our quest to further improve one of our most important crops through molecular breeding. References Fouquet R, et al. 2011. Maize Rough Endosperm3 Encodes an RNA Splicing Factor Required for Endosperm Cell Differentiation and Has a Nonautonomous Effect on Embryo Development. Plant Cell 23: 4280-4297. Fu S, Meeley R, Scanlon MJ 2002. empty pericarp2 Encodes a Negative Regulator of the Heat Shock Response and Is Required for Maize Embryogenesis. Plant Cell 14: 3119–3132. Hurov KE, Cotta-Ramusino C, Elledge SJ 2010. A genetic screen identifies the Triple T complex required for DNA damage signaling and ATM and ATR stability. Genes Dev 24: 1939-1950. Kynast RG, et al. 2001. A Complete Set of Maize Individual Chromosome Additions to the Oat Genome. Plant Physiology 125: 1216-1227. Li Y, Segal G, Wang Q, Dooner HK. 2013. Gene tagging with engineered Ds elements in maize. In: Peterson T, editor. Plant Transposable Elements: Methods and Protocols: Humana Press. p. 83-99. Lid SE, et al. 2002. The defective kernel 1 (dek1) gene required for aleurone cell development in the endosperm of maize grains encodes a membrane protein of the calpain gene superfamily. Proc Natl Acad Sci U S A 99: 5460-5465. Mertz ET, Bates LS, Nelson OE 1964. Mutant gene that changes protein composition and increases lysine content of maize endosperm. Science 145: 279–280. Messing J, Fisher H 1991. Maternal effect on high methionine levels in hybrid corn. Journal of Biotechnology 21: 229-237. Rines HW, et al. 2009. Addition of individual chromosomes of maize inbreds B73 and Mo17 to oat cultivars Starter and Sun II: maize chromosome retention, transmission, and plant phenotype. Theor Appl Genet 119: 1255-1264. Takai H, Xie Y, de Lange T, Pavletich NP 2010. Tel2 structure and function in the Hsp90-dependent maturation of mTOR and ATR complexes. Genes Dev 24: 2019-2030. doi: 10.1101/gad.1956410 Udy DB, Belcher S, Williams-Carrier S, Gualberto JM, Barkan A 2012. Effects of Reduced Chloroplast Gene Copy Number on Chloroplast Gene Expression in Maize. Plant Physiology 160: 1420-1431. Wang G, et al. 2014. Proline responding1 Plays a Critical Role in Regulating General Protein Synthesis and the Cell Cycle in Maize. Plant Cell 26: 2582-2600. White PJ, Weber EJ. 2003. Lipids of the kernel. In: White PJ, Johnson LA, editors. Corn: Chemistry and Technology. St. Paul, Minnesota: American Association of Cereal Chemists, Inc. Wu Y, Holding DR, Messing J 2010. Gamma-Zeins are essential for endosperm modification in quality protein maize. Proc Natl Acad Sci U S A 107: 12810–12815. Xu JH, Messing J 2009. Amplification of prolamin storage protein genes in different subfamilies of the Poaceae. Theor Appl Genet 119: 1397-1412.

ETD_8076

Ph.D.

Includes bibliographical references

by Nelson S. Garcia

doi:10.7282/T3M048B0

ETD doctoral

The author owns the copyright to this work.