IDENTIFICATION AND ANALYSIS OF NEW STARCH METABOLIC MUTANTS IN ARABIDOPSIS THALIANA

Abstract

Starch is the primary carbohydrate reserve in plants and an important source of nutrition for humans, which is synthesized and stored in the form of water-insoluble, semi-crystalline granules. Storage starch accumulates in the amyloplasts of seeds and tubers and plays an important role in long-term energy storage for the use in germination and early plant development. In addition, in chloroplasts of leaves, transitory starch is produced during the day using photoassimilates and is subsequently degraded at night to sustain metabolic processes when photosynthesis is not possible. Storage starch and transitory starch are similar in structure, but while storage starch granules display a wide range of shapes and sizes, transitory starch granules typically exhibit a consistent morphology across different species - they are usually lenticular in shape and measure a few micrometers in diameter. Although considerable progress has been made in understanding the biosynthesis and degradation of starch, the initial stages of starch granule formation, which influence granule number and morphology, remain less understood. In recent years, several proteins have been discovered that form a complex network of protein-protein interactions and are required for granule initiation. However, there may be alternative pathways involving other proteins at play. In addition, very little is known about the factors leading to the lenticular shape of granules. In this study, I conducted a forward genetic screen to identify novel genetic factors that influence the size and shape of chloroplast granules. This screen used flow cytometry as a high-throughput method to analyze the starch granule morphologies from a mutagenized Arabidopsis plant population. In Chapter 1 of my thesis, I tested the applicability of flow cytometry to analyze the size and shape of starch granules in Arabidopsis leaves, along with the analysis of known starch metabolism mutants. My investigations showed that the known starch granule initiation proteins were the most influential factors, as mutants lacking any one of them displayed obvious alterations in granule morphology. However, several mutants of proteins involved in starch biosynthesis, crystallization, and degradation also consistently showed alterations in granule morphology, some of which had previously been overlooked. This demonstrated the sensitivity and robustness of flow cytometry to quantify even subtle granule differences. Using flow cytometry for my forward genetic screen allowed me to identify new alleles of almost all genes anticipated to be identified, providing new insights into the functions and importance of their protein domains. Importantly, I also discovered three mutants of genes, whose involvement in starch morphogenesis was previously unknown. Chapter 2 is dedicated to the analysis of one of these novel mutants, psbw-2. This mutant had a glycine-to-arginine conversion in the transmembrane region of PsbW, a protein integral to thylakoids and involved in photosystem II complex formation. The resulting PsbWG107R protein is still integral to the membranes, but its insertion causes severe distortion of the thylakoid membranes in a dosage-dependent manner. This was accompanied by the formation of numerous small, irregularly shaped granules in the chloroplasts - a phenotype strikingly different from that of the psbw-1 knockout, which showed no alterations in membrane organization or granule formation. These data suggest that PsbWG107R has gained an aberrant function, with the presence of arginine in its transmembrane helix possibly leading to membrane distortion to accommodate the positive charge. It also supports emerging evidence that thylakoids control granule formation in multiple ways, providing a new perspective on how they help determine granule shape and number. Detailed analyses of the remaining two novel factors identified in my screen may further improve our understanding of the mechanisms controlling granule number and shape.