Plants are generally unable to move reasonable distances as single adult organisms. This lack of mobility necessitates robust pathways that allow for response to environmental stimuli which can cause changes in the plant’s morphology to adapt to their changing environment. One such trait is flowering, and it is crucial for plant survival because it allows for plants to reproduce. The vast majority of agricultural plants are flowering plants, and robust growth and reproduction of these plants is especially important for the growing human population. This experiment aims to understand the interaction between an Arabidopsis thaliana nitrogen response gene named NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1), and an Arabidopsis gene which controls flowering called FLOWERING LOCUS T (FT). NIGT1 is known to modulate plant flowering despite being a nitrogen response gene. Here, I used a Dual-Luciferase Reporter System to test whether NIGT1 proteins directly interact with the FT promoter to regulate FT gene expression. In this system, the FT promoter is used to drive expression of Firefly Luciferase rather than FT. Separately, the NIGT1 gene coding region will be highly expressed under a constitutive 35S promoter. It will also be fused to the transactivation domain of the viral protein VP16 which converts transcriptional repression into transcriptional activation, as NIGT1 is known to be a transcriptional repressor. The VP16 transactivation domain will be repeated as four tandem repeats, forming the construct called VP64. The gene constructs of interest will be inserted into Nicotiana benthamiana using agrobacterium infiltration, then the leaf material from N. benthamiana will be used for the Luciferase assay. I hypothesize that the binding of NIGT-VP64 to the FT promoter will cause increased in Firefly Luciferase production compared to the absence of NIGT1-VP64. Higher amounts of Firefly Luciferase will result in greater luminescence, which we will quantify with a luminometer.

Eelgrass (Zostera marina) is a marine flowering plant that provides important ecological, environmental, and economic services in the Puget Sound. Z. marina reproduces both asexually and sexually; however, the cellular regulation behind each mode of reproduction is poorly understood. Establishing Z. marina’s regulation behind sexual reproduction will improve predictions of Z. marina’s resilience to anthropogenic pressures. FLOWERING LOCUS (FT) is a highly conserved gene that promotes floral development in flowering plants and is well-characterized in the model organism A. thaliana. As a marine flowering plant, we hypothesized that a homologous, functionall FT exists within Z. marina. We identified 13 candidate Z. marina FT homologs (ZmFT), and from analysis of relative flowering time, 5 homologs of interest emerged: 2 that promote flowering, 2 that inhibit flowering, and 1 that has striking sequence conservation to A. thaliana FT, yet has no observable impact on flowering. To determine how intracellular distinctions alter the function of these respective Z. marina FT homologs, I am currently investigating putative protein-protein interactions of the ZmFT homologs with components of the A. thaliana floral activating complex, such floral promoting transcription factors, and scaffold proteins . I can characterize these protein-protein interactions using Yeast Two-Hybrid assays (Y2H) and BiFC fluorescent microscopy In which protein-protein interactions between ZmFT and A.thaliana floral complex proteins yields either yeast strains with conferred prototrophy, in Y2H, or a fluorescent signal, in BiFC. From my Y2H and BiFC work, I will characterize interactions of the ZmFT homolog proteins, with known components of the A. thaliana flowering pathway. The results of this study will help define how Z. marina regulates flowering onset and sexual reproduction.