Plants’ ability to sense ambient light conditions is critical for their fitness. Phytochromes (phy) are photoreceptors that translocate to the nucleus when activated by red light. One consequence of this translocation is rapid phosphorylation and turnover of basic helix-loop-helix (bHLH) transcription factors called Phytochrome-interacting factors (PIF). PhyB has been shown to recruit and inactivate PIF5 in seedlings and inhibit elongation of the stem (hypocotyl). Phytochrome mutants in Arabidopsis thaliana exhibit reduced CO₂ uptake, abnormal accumulation of sugars and metabolites during the day, and shade avoidance-like phenotypes. We aim to compare findings in A. thaliana with phenotypes of light signaling mutants in Brassica rapa, a related crop species. We have isolated phyB, and pif5 mutants and are growing them in ambient and high CO₂ conditions. Measurements of photosynthetic rate as well as levels of chlorophyll, sugars, total protein and starch will provide insights into the role of Phytochrome signaling on metabolic processes. Preliminary results show that phyB mutant seedlings exhibit longer hypocotyls in comparison to wild-type plants at ambient CO₂ but have a reduced growth response to elevated CO₂ compared to wild-type. Adult plants have a reduced photosynthetic rate compared to wild-type. In contrast, pif5 mutant seedlings have short stems and reduced root growth in comparison to wild type. Our results to date are consistent with previous studies in A. thaliana. Examining the role phytochromes play in resource allocation, specifically in response to Carbon availability will aid in identifying key phytochrome-mediated developmental pathways. This research has implications in engineering more efficient crops and in improving understanding of plant metabolism as a whole.

Pacific cod (Gadus macrocephalus) is the target species of a large fishery in North America that ranges from Alaska to California. In a recent genetic sequencing study, very high divergence among Pacific cod from different geographic areas was observed in what appears to be the Zona pellucida sperm-binding protein 3 (ZP3) gene. In many other vertebrates, the ZP3 gene codes for proteins that are significant to reproduction, and this gene may be an important factor in the context of the species' life history and management. To increase resolution of how ZP3 varies across the Pacific cod range, this project investigates the entire coding sequence in Pacific cod from four geographic areas: Kodiak Island, Prince Williams Sound, Hecate Strait, and the Washington Coast using PCR and Sanger sequencing. Differences in amino acids and protein structure were identified. It appears that there are significant differences in the ZP3 gene between southern and northern populations of Pacific cod. Understanding these patterns is important for future studies of how differences in the ZP3 gene may affect reproduction and population connectivity.

The plant hormone auxin controls nearly every aspect of plant growth and development. Proteins involved in the perception of auxin are closely studied in an effort to elucidate drivers of plant morphology. Potential applications of such research lie in engineering of plant architecture and ultimately maximizing crop yield. Auxin triggers binding between two proteins: auxin receptors (Auxin-signaling F-Boxes or AFBs) and transcriptional repressors (Aux/IAAs). This interaction leads to the ligation of ubiquitin to Aux/IAAs, triggering their degradation, and allowing transcription of auxin-induced genes. In the model plant Arabidopsis thaliana, the AFB family has six members (TIR1 and AFB1- AFB5). While the AFBs are highly similar in sequence to one another, individual AFBs may play specialized roles. In our preliminary studies, we have found that a reduction in AFB2 activity in the afb2-3 mutant produces a leaf phenotype not observed in TIR1 mutants. To confirm that AFB2 is responsible for this phenotype, we are engineering transgenic afb2 mutants that express a wild-type copy of AFB2. We expect that leaves of these transgenic plants will resemble those of wild-type. We are also analyzing the leaf phenotypes of other AFB mutants. To test whether small differences in AFB2 function can change leaf phenotype, we are currently transforming natural hypomorphic and hypermorphic AFB2 variants into afb2-3 mutants. By quantifying leaf morphology of these transgenic plants, we plan to map the relationship between AFB2 function and leaf phenotype.

Crop production will have to double by 2050 to meet projected global food demands. Insect herbivory currently accounts for nearly 15% of global crop losses; thus, plants better able to repel insect attack should have a significant impact on food security. One successful strategy for engineering plants is the constitutive expression of insecticidal toxins such as Bacillus thuringiensis endotoxin (BT). However, the constant production of these toxins decreases crop yield and increases the development of BT-resistant pests. When plants are damaged by insects, they produce the phytohormone jasmonate. We propose a new plant engineering strategy that links the expression of BT to jasmonate signaling. To create this link we have built a synthetic transcription factor called a Jasmonate Degradable CRISPR Transcription Factor (JDCTF). This transcription factor is easily retargeted to any gene of interest by changing the dcas9-associated guide RNA. The JDCTF responds to jasmonate via a jasmonate sensitive degradation domain. Using the plant model Arabidopsis thaliana, we’ve generated transgenic plant lines expressing a JDCTF that targets a Venus-Luciferase reporter. We have shown the system works using a bioluminescence time course assay on seedlings treated with the jasmonate analog coronatine. After induction with coronatine, we observed an increase in bioluminescence in the treated seedlings and no increase in biolumescence for the control. Currently we are characterizing the dynamic response to both exogenous jasmonate, as well as simulated and real insect herbivory on mature leaves. Future work will focus on regulating BT expression with the JDCTF system. Our system has the potential to increase yields, increase consumer safety, and slow down the development of insecticide resistance in pests.

Climate change is threatening global food security. There is a pressing need for better crops that can prevent severe losses. We hope to apply our current understanding of plant developmental mechanisms that are involved in responses to harsh external stimuli to engineer more resistant crop varieties. The plant hormone auxin plays an important role in organogenesis, the process of new organ formation. Auxin controls organogenesis by accumulating in specific tissues at specific developmental times. These accumulations are due to the polar flow of auxin through the plant. These flows are in turn reinforced by auxin through a feedback mechanism called canalization. PIN1 is a an auxin efflux transporter that is required for polar auxin transport. Loss of PIN1 results in several developmental defects. These phenotypes and other analyses have led to mathematical models of organogenesis. We used these models to predict plant phenotype in response to increased canalization. We have engineered transgenic plants with a library of synthetic promoters with varying auxin sensitivity driving expression of PIN1. We are characterizing the shoot architectures of these plants, specifically the number of branches and the pattern of lateral organ initiation. We are also studying how the increased canalization affects the distribution of auxin in our plant lines using fluorescent reporters. We hope that our results will demonstrate how plant synthetic biology and predictive models can be used to rationally design plant developmental phenotypes.

Brassinosteroids and auxin are plant hormones that are crucial in plant growth development. Although it is known that brassinosteroids and auxin signaling interact, it is not known exactly how. We are focusing our attention on the question of how auxin and brassinosteroids interact during the formation of lateral roots. Lateral roots are roots that extend horizontally from the primary root and the developmental tradeoff between the primary and lateral root formation helps the plant optimize its water and nutrient uptake. Previous studies have demonstrated that auxin promotes specification of founder cells, the first step in making a new root. To test if brassinosteroids are also involved in this process, I am quantifying the density of founder cells in seedlings treated with brassinosteroids alone, auxin alone or both hormones together. In this study, I will be able to distinguish whether the hormones act additively or synergistically (a much greater effect than a simply addition of the effect of each hormone individually). Our study will deepen our understanding of root formation which will have agricultural significance as strong roots lead to effective nutrient and water intake and stability for the plant under varying weather conditions.

In recent decades, coral reef ecosystems have experienced mass mortality events related to increased sea surface temperatures and coral bleaching. Although numerous studies have documented the loss of coral diversity and abundance after bleaching, less is known about the potential of these communities to recover over longer time periods. We investigated whether reef communities in Bocas del Toro, Panama, were able to recover from two bleaching events: a moderate event in 2005 and a severe event in 2010. Ten reefs were randomly sampled using underwater photography before bleaching occurred (2004) and four years post-bleaching (2013). We identified marine organisms (corals, sea urchins, sponges, algae, etc.) and substrate type for 3,500 random points across a depth gradient on each reef. Subsequently, we investigated trends in reef community composition across the two different sampling periods. We hypothesize that coral recovery will be influenced by different life history strategies such as reproductive behavior and tolerance to environmental perturbation. Preliminary data suggest that sea urchins have increased in abundance since 2004, while algal cover has declined. We hope that this study will provide more information on the contribution of invertebrate herbivores to reef recovery and resilience.