Many plant-pollinator interactions are thought to depend on coordination between the timing of plant scent emission and pollinator activity. Certain plants of the genus Petunia exhibit rhythmic scent emission based on the circadian clock, but little is known about the role of the clock in pollinator behavior. The pollinator of Petunia axillaris, the hawkmoth Manduca sexta, is thought to be nocturnal based on mostly anecdotal evidence. To understand the temporal relationship between Manduca sexta and P. axillaris, it is necessary to first determine the precise timing of hawkmoth activity. We hypothesized that moths would display circadian timing in their flight behavior, complementing the timing of scent emission from P. axillaris. To test this, we observed the flight behavior of male hawkmoths in response to 12-hour light/12-hour dark conditions, continuous dark, or continuous light. The moths were entrained to the 12-hour light/12-hour dark cycle during their pupation and their first two days post-cocoon emergence, and they were introduced to a wind tunnel on the third day for the experimental conditions. The number of moths flying within a ten-minute period was recorded continuously for one day or every four hours for three days. Hawkmoths exhibited flight activity only during the subjective night phase of continuous dark conditions. This rhythm was maintained over multiple days of continuous darkness, demonstrating that it is not dependent on light input. We also examine how desynchronizing the clocks between plant and pollinator affects pollination success, and lastly if it’s possible to induce a pollinator shift by shifting the timing of scent release.

Coordination of the initiation of flowering with the optimum season is important for plants in both natural and agricultural systems. In the model plant, Arabidopsis thaliana, timing of flowering is strongly regulated by photoperiod. The main factors important for flowering are CONSTANS (CO) protein, which is an activator of flowering in response to photoperiod and FLOWERING LOCUS T (FT), the direct target of CO. FT protein is a mobile factor which can initiate flowering. Additionally there is a family of repressor transcription factors, the CYCLING DOF FACTOR (CDF) transcription factors that are able to limit the timing of both CO and FT expression so that flowering is repressed during short days. Functionally, CDF expression is controlled by the circadian clock so that their effect is constrained to the morning period of the day. In the afternoon of long days, CDF protein is degraded, enabling CO and FT to promote flowering. Recently, our laboratory has determined that CDFs may require a co-repressor protein, TOPLESS (TPL) to form a repressor complex to regulate flowering genes. To explore the importance of this repressor complex on flowering time, I observed how FT, CO, and other flowering genes expression levels change over the course of development in several genetic backgrounds. To remove TPL function where flowering time genes are expressed, we created a transgenic plant that expresses a dominant negative mutant of TPL in phloem companion cells. We hypothesized that this mutant would resemble a CDF loss of function, so I compared these lines and wild type plants over developmental time. Additionally, I am investigating the genetic interactions and epistasis of TPL with other flowering time pathway components by checking the flowering time and gene expression of transgenic plants which express mutated TPL and harbor mutations in other flowering time genes.

Flowering time in plants is imperative to reproductive viability and is controlled by a variety of molecular regulatory systems drawing upon internal and external stimulus. We use Arabadopsis thaliana as a model to determine how plants incorporate photoperiodic sunlight cues in the production of flowers. Through the transcriptional regulation of certain molecular factors, plants are able to effectively control the temporal expression of the FT (FLOWERING LOCUS T) gene, FT protein is in leaves and then moves up the shoot apex to initiate flowering. Expression of the FT gene is initiated by a transcription factor called CO (CONSTANS) and suppressed by a protein called CDF1 (CYCLING DOF FACTOR 1) that are controlled by sunlight and the plant’s endogenous circadian clock. CDF1 inhibits CO transcription by binding to a series of four identical upstream DNA binding sites and is thought to do so in conjunction with a corepressor protein called TPL (TOPLESS). In this experiment we created a mutant background that was deficient in TPL activity in conjunction with a synthetic CO promoter fused to the bioluminescent enzyme firefly luciferase (LUC). We use this tool to assay if changes in binding site number and orientation in the presence or absence of TPL change the sensitivity of the promoter to photoperiodic sunlight inputs. We hypothesize that the removal of TPL will increase early-day CO expression regardless of daylength but CO expression will be unaffected by the amount of binding sites. We also expect that loss of TPL function attenuates CDF1 dependent repression of flowering, such that differences in CDF1 repression between different binding site architectures types will be unchanged in the absence of the corepressor. This would corroborate results from previous experiments. Should we obtain other results we might be made to reevaluate the role TPL plays in florigenesis in Arabidopsis.