Molecular mechanisms that regulate the amino acid composition in Arabidopsis seeds
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Amino acids (AA) are the building blocks of proteins, which makes them extremely important for growth and development. Crop seeds, such as legumes and cereals, play an essential role as a key food source in the diet of humans and livestock but do not meet the dietary requirements of essential amino acids (EAA), which are the AA that humans and vertebrates cannot synthesize and must obtain from the diet. Lacking sufficient levels of EAA in the diet can lead to protein-energy malnutrition, which adversely affects the immune, gastrointestinal, nervous, and cardiovascular systems. Efforts to fortify AA composition in crop seeds have had a very limited success because plants respond to induced protein composition alterations by activating a regulatory mechanism that "resets" it back to the original state. This phenomenon is known as proteome re-balancing, and, while beneficial for plants' growth and development, has been a major hurdle to biofortification. A good understanding of the regulation and rebalancing mechanism of AA in seeds would improve the biofortification of AA composition. Chapter One of this dissertation provides a comprehensive introduction from previous studies of what is known about amino acid composition in seeds, the challenges identified in previous experimentation, and how the content of the other chapters builds upon and adds value to the area of seed amino acid research and to the understanding of proteome rebalancing. Chapter Two uncovers the genes and biological processes that underly proteome relancing using mutants of the most abundant seed storage proteins (SSPs), the cruciferins also known as 12S. To better understand how proteome rebalancing is achieved in seeds, we conducted a comprehensive analysis on Arabidopsis mutants lacking the three most abundant SSPs, the cruciferins (CRUs). The multi-omics analysis such as transcriptome, proteome, metabolome, and measurement of physiology parameters was conducted on single mutants (crua, crub, and cruc) and the triple knock out (cruabc) mutants compared to the wild type (Col-0). All major seeds storage compounds remained unchanged in these mutants suggesting rebalanced seeds. Further analysis showed that translation and oxidative stress responses are the two key biological processes that dominated proteome rebalancing. Chapter Three focuses on how proteome rebalancing is achieved in the second most abundant seed storage proteins known as the Napins or 2S. In this chapter, I used one napin-RNAi (RNA interference) to target all 5 members of this gene family. Using the multi-omics analyses, all reserve compounds remained the same compared to Col-0 except the sulfur. Only oxidative stress response dominated the biological processes involved in proteome rebalancing in the Napin seed storage proteins. Chapter Four covers the remaining seed storage protein mutants from the cruciferins, these were the double mutants (cruab, cruac, and crubc). In this chapter, all analyses performed compared the doubles (cruab, cruac, and crubc) to Col-0; genes and key pathways involved in proteome rebalancing were revealed. Lastly, Chapter Five, the conclusion, reiterates the contributions of this dissertation to the field of seed amino acid proteome rebalancing and provides future directions and research projects that can be done to bring much more insight into this field.
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Ph. D.