Eelgrass (Zostera marina) is a crucial marine flowering plant that provides intertidal ecosystem services in and outside the Puget Sound. However, eelgrass populations are under threat due to the adverse effects of climate change. Maintaining genetic diversity is essential for their survival. This project aims to enhance our understanding of eelgrass reproduction and how populations will react to climate change by characterizing the molecular mechanisms of flowering in eelgrass. We have identified candidate eelgrass flowering genes that are homologous to genes found in Arabidopsis thaliana, a model organism. These candidate genes were selected based on the clock-regulated model of flowering, in which FLOWERING LOCUS T (FT) encodes for a critical protein that triggers the flowering response pathway. To investigate this model, we created transgenic overexpression lines in Arabidopsis and identified two candidate eelgrass FT genes that exhibit an early flowering phenotype similar to FT overexpression in Arabidopsis. We are also using Yeast 2-Hybrid Assays (Y2H) to confirm the identity of the suspected eelgrass FT. Y2H is a technique for studying protein-protein interactions and allows us to investigate interactions between eelgrass FT and other transcriptional regulators within the flowering response pathway. Based on our data, we expect to see the two candidate eelgrass FT genes result in a simultaneous early flowering phenotype and in interactions with other transcriptional regulators to form a transcription factor complex that enables the expression of a reporter gene. Identifying FT in eelgrass will prompt further study of individual and population-wide flowering patterns, and how these patterns change with shifting ocean conditions.

One of the most striking adaptations in land plant evolution is the specialization of reproductive structures. LEAFY (LFY) is well characterized in flowering plants (angiosperms) as a transcription factor initiating the development of the flower, which contains the reproductive organs. LFY also regulates the first cell division of the zygote (diploid phase) in mosses (a type of non-vascular plant). The fern Ceratopteris richardii is a type of non-flowering plant that is midway phylogenetically between mosses and flowering plants, in the sister clade to seed plants. With a lab-friendly, independent haploid phase (gametophyte), transgenic protocols and a reference genome, C. richardii is ideal for studying LFY’s functional evolution. Previously, targeted silencing of the two fern LFY homologs CrLFY1/2, demonstrated that it maintains the identity of the apical stem cell in gametophytes. To further characterize the function of fern LFY, I study the effects of its over expression on gametophyte development. To that end, I record gametophyte development using bright-field and fluorescent microscopy. Preliminary results suggest that overexpression of CrLFY may affect development of the sperm-producing gametangia (antheridia) in fern gametophytes, with more antheridia found in transgenic plants late in development. Given that antheridia continue to be produced in wild type gametophytes in the absence of fertilization, I test the hypothesis that CrLFY overexpression causes delayed fertilization (by a yet unknown mechanism) and that increased antheridia represent a secondary effect. Here, I experimentally delay fertilization by withholding water needed for sperm release (“flooding”), and compare the number and pattern of antheridia on transgenic and wild type gametophytes with and without flooding. Functional characterization of LEAFY in a fern, and of other master regulators of development more generally, contributes to a better understanding of the evolution of land plants via the potential repurposing of ancestral genetic pathways into novel functions.

Climate change and global population growth are driving the need for more sustainable methods for growing crops used in agriculture and the production of biofuels. To address these challenges we are exploring the role of that plant microbiome in host plant tolerance to environmental stresses related to climate change. In particular we are investigating whether endophytes, microorganisms that colonize the internal tissues of plants, make the host plants more tolerant to drought or low-nitrogen conditions. We are currently optimizing the process of DNA extractions of fruit and poplar trees which were inoculated with beneficial nitrogen fixing bacteria, and grown in either water-or nitrogen limited conditions. We then purify high quality microbial DNA, and use polymerase-chain reaction (PCR) to optimize strain specific primers (SSP), which target specific DNA sequences in the genomes of our endophytes, and minimize background noise produced by plant and other microbial DNA. This allows us to reisolate our strains from the plants to gain understanding of where they colonize, and to demonstrate that the trees were successfully colonized by our endophytes to support growth and drought tolerance data collected from inoculated and uninoculated controls. By ensuring the SSPs only target our strains of interest, we differentiate our endophytes from other members of the plant microbiome. These primers are then used in Droplet Digital PCR (ddPCR) to quantify their relative abundance. Using this information we hope to contribute to the project objectives of demonstrating that beneficial endophytes can be used as a sustainable method for improving drought and low nitrogen tolerance in plants, both in agricultural and biofuel applications, reducing the consumption of nitrogen fertilizers and water for irrigation in these sectors.

Wetland ecosystems are in peril as they are becoming increasingly smaller and more fragmented, due to an increase in land being converted for agricultural and industrial purposes. This fragmentation increases the importance of effective seed dispersal for wetland grasses, which can be the determining factor for the survival of species on a regional level. Grasses are a fundamental component of wetland habitats, yet much remains unknown in relation to their seed dispersal strategies. Studying seed dispersal methods can provide insight on the restoration potential of threatened wetland ecosystems and species, and help inform conservation and management decisions. My research aims to discover the morphological traits that facilitate effective seed dispersal in wetland grasses, and the factors influencing the evolution of these traits. I hypothesize that grasses restricted to wetland habitats have dispersal morphology that is highly specialized, while facultative and non-wetland species tend to use more generalist dispersal strategies, assisting dispersal under a wider range of conditions. To test this hypothesis, I sampled 81 grass species from wetland genera and measured morphological seed traits (eg., falling velocity, mass) which are known to facilitate certain dispersal methods (eg., wind or water dispersed). These data were analyzed by constructing a multi-dimensional ecomorphospace to visualize the range of dispersal morphologies in wetland and non-wetland grasses. Preliminary results suggest that wetland grass seeds are less capable of wind dispersal than their non-wetland relatives, yet there is no difference in seed mass between wetland grasses and their non-wetland relatives. This research holds conservation implications as understanding the conditions that drive grasses to adopt one dispersal strategy over another can be used to forecast the future of wetland grass species and the future health of wetland ecosystems overall.

Hydrogen sulfide (H₂S) impacts biological systems in multiple ways, including the arrest of aerobic respiration, and thus is mechanistically similar to cyanide. Unlike cyanide, however, H₂S can accelerate as well as end cell growth, including in plants, where it drives germination rates when administered in micromolar concentrations. However, the limited research to date leads to a need to better quantify and contextualize chemical composition changes in plant tissue following H₂S-induced plant growth. This study resulted in the ability to quantify the biophysical impacts of H₂S-induced growth in plants. The novel use of δ³⁴S and δ¹⁵N isotope ratios produced at the IsoLab in University of Washington represent the results of this sampling and its subsequent analysis. They may represent a new means to understanding the effects of H₂S on plant growth, including during the crucial phase of plant germination. The effects were observed on hypocotyl tissues from seedlings of Pisum sativum (pea), Phaseolus vulgaris (bean), and Zea mays L. (corn), all grown in hydroponic H₂S solutions, ranging from 0-100μM. These specific isotopic methods may allow comparison between modern and fossil material, because these isotopic species are known to have been preserved across a wide diversity of plant fossils. This novel application of these classic staples in the biochemical toolbox may have further implications for better understanding past events, because many major mass extinctions have now been linked to excess oceanic and atmospheric H₂S (compared to today), and may also, paradoxically, present new paths toward increased crop yields.

 Reconstructing past environments can help us understand plant community evolution over time. For example, plant silica (phytoliths) can help us reconstruct canopy openness. Phytoliths are formed when plants uptake monosilicic acid from the surrounding soil through their roots and deposit it as opalized silica in and around cells; they have been used as a tool in paleoecology because they are well-preserved in the fossil record. For phytoliths formed in the outermost layers of leaves (epidermis), there is a relationship between morphology and light availability. A previous method established this correlation using modern soils in Costa Rica to apply to sites in the Eocene-Miocene of Argentina. However, it is unclear whether this model can make accurate inferences in other geographic regions. Here, we expand the method using modern phytolith samples from the Southeastern United States to generate a dataset and apply it to fossil phytolith assemblages from the North American Great Plains Region to reconstruct changes in vegetation during Oligocene-Miocene grassland expansion. For this work, we use an optical microscope to observe and count the phytolith assemblages to reflect a range of vegetation types in North America. We focus on phytolith morphotypes representing silicified epidermal pavement cells and measure their size and shape using ImageJ. We expect a linear trend between LAI (Leaf Area Index, the quantified relationship between morphology and light availability) from phytoliths and observed LAI which can be used to form the model for North American environments and applied to the fossil phytolith record of the Great Plains Region. Expanding on this method could make its use more widespread and lead to similar research in other regions of the world. Current models suggest the persistence of closed forests through this entire interval, a result we wish to further test using this updated model.