Electronic Thesis and Dissertation Repository


Doctor of Philosophy




Dr. Rima Menassa


During seed development, the lytic vacuole (LV) is replaced by a protein storage vacuole (PSV) which specializes in accumulating seed storage proteins (SSPs). As seed protein reserves are mobilized upon germination, the PSV is once again replaced by the LV which takes on different roles in vegetative tissues. Cellular events occurring during these developmental transitions are not well understood, particularly, the transition between vacuole types. This research investigates whether PSVs can exist in leaves. To study vacuole transitions in leaves, an Arabidopsis thaliana line over-expressing the LEAFY COTYLEDON2 (LEC2) transcription factor was used. LEC2 is a master regulator of embryogenesis responsible for creating a cellular environment that promotes embryogenic development. Over-expression of LEC2 causes vegetative tissues to change their developmental fate to an embryonic state. LEC2 alters the leaf phenotype at the subcellular level; chloroplasts de-differentiated and contained more starch. The cytoplasm becomes filled with oil bodies, which are typically seed organelles. The large LV was replaced by small-sized vacuoles that accumulated protein deposits. Since LEC2 is responsible for activating the synthesis of SSPs during seed development, SSP accumulation was investigated in leaves. The major Arabidopsis SSP families were shown to accumulate within small sized vacuoles in leaf cells. By exploiting the developmental and tissue specific localization of two tonoplast intrinsic protein (TIP) isoforms, the small vacuoles were identified as PSVs. A time course following the cellular alterations and accumulation of seed proteins in leaves after induction of LEC2 activity with dexamethasone (DEX) revealed the appearance of embryonic characteristics as early as 4 days on DEX and became more prominent over time. Additionally, a dynamic view of the transition between vacuole types was observed using TIP isoforms fused to fluorescent markers. The morphology of leaf vacuoles was altered to resemble an amalgamation of a LV and PSV. Results suggest that as the LV transitions to a PSV, the tonoplast remodels before the large vacuole is replaced by smaller PSVs. The formation of PSVs in leaves in response to LEC2 over-expression is a novel approach to study vacuoles and will lead to a better understanding of their basic biology.

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