Date of Award

12-2015

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Legacy Department

Genetics

Advisor

Luo, Hong

Committee Member

Luo, Hong

Committee Member

Marcotte, William

Committee Member

Frugoli, Julia

Committee Member

Wang, Liangjiang

Abstract

World population will pass nine billion by 2050, while the agricultural land area will not increase dramatically in the coming decades. To meet the ever-increasing food demand, genetically engineered crops have been rapidly adopted for crop productivity. MicroRNAs have become increasingly attractive as targets in crop genetic modification due to their regulatory role in fine-tuning many essential biological processes. My research explores the potentials of microRNA528 (miR528) and miR396 for use in genetic modifications of the important agronomic traits of plant development, abiotic stress response, and/or flowering time control in an economically and environmentally important perennial monocot species, creeping bentgrass (Agrostis stolonifera). MiR528 is a conserved monocot-specific small RNA which is involved in multiple stress responses, however, experimental functional studies are lacking. In this study, we generated transgenic creeping bentgrass overexpressing a rice miRNA, Osa-miR528. Transgenic plants show altered plant development and enhanced salt and nitrogen (N) deficiency tolerance. The developmental changes include shortened internodes, increased tillers, and relatively upright growth. Enhanced salt stress tolerance is associated with improved water, chlorophyll, and potassium retention, cell membrane integrity, increased proline content, CATALASE activity, and reduced ASCORBIC ACID OXIDASE (AAO) activity; while improved N deficiency is associated with increased biomass, total N accumulation, chlorophyll synthesis, nitrite reductase activity, and reduced AAO activity. Molecular analysis identified AAO and COPPER ION BINDING1 as putative targets of miR528 in creeping bentgrass. Both putative targets respond to salt stress and N deficiency. The conserved miR396 is involved in plant vegetative and reproductive development and various environmental stress responses through targeting Growth Regulating Factor (GRF) transcription factor genes. In this study, we generated transgenic creeping bentgrass overexpressing a rice miRNA, Osa-miR396c. The transgenic plants exhibit altered development in both vegetative and reproductive growth, elimination of vernalization requirement, and enhanced salt stress tolerance. The altered plant development includes less shoot and root biomass, shorter internodes, smaller leaf area, fewer leaf veins and epidermal cells, and defects in filament elongation, anther dehiscence, and pollen viability. The enhanced salt tolerance is associated with improved water and chlorophyll retention, cell membrane integrity, and sodium ion exclusion during salt stress. The elimination of vernalization requirement is associated with the regulation of key genes VRN1, VRN2, and VRN3 in the vernalization pathway. Four putative targets AsGRF3-6 were identified in creeping bentgrass. These targets respond to high salinity, long-day photoperiod, and prolonged cold exposure. RNA-seq analysis shows that differentially expressed genes are involved in cell division, floral organ development, vegetative to reproductive transition, histone modification, oxidation reduction, environmental stress response, etc. The study established the molecular pathways of miR528- and miR396-mediated plant salt stress and/or N deficiency tolerance in creeping bentgrass. It provides insight into miRNA-mediated regulatory network in plant vegetative and reproductive development, abiotic stress response and flowering time control.

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