Castilleja levisecta (CALE) is a threatened, hemiparasitic angiosperm, endemic to Pacific Northwest prairie ecosystems. Like other parasitic plants, CALE forms haustoria: root structures that connect the xylems of the two plants together. CALE then uses this connection to steal nutrients from the host plant to assist its own growth, without killing the host. While it is known that CALE will form haustorial connections with a number of host species, the quantities and timing of specific nutrients taken from the host plants remains a mystery. How old does CALE have to be before optimum nutrient uptake occurs? How does the chemical profile of the host plant change CALE’s growth rate and chemical profile, and are there tertiary effects on pollinators and herbivores that use CALE as their food source or habitat? We cannot begin to answer these questions until we know the basics of nutrient transfer from host to CALE. In this pilot study, we have labeled host plants with heavy isotopes of nitrogen and carbon, to track the length of time it takes CALE to acquire those nutrients via the haustoria, and the amount of each nutrient acquired from the host. We planted over 70 pairs of CALE and Eriophyllum lanatum (ERLA), one of CALE’s known host species. The roots of each seedling were planted across each other, to encourage haustorial connections to form. After several weeks of growth in pairs, heavy isotopes were applied to ERLA with the expectation that they would be taken up by CALE. Both plants were later dried and ground up for analysis of heavy isotope content. Analysis of these data will give us a better understanding of the timing and quantity of host nutrient uptake by CALE. This information will be used to inform efforts to restore and conserve populations of this threatened species.

Our study aims to prove that adequate algal biofuel production is feasible in the Pacific Northwest and expand the reach of renewable energy. Global energy demand is increasing along with the interest in renewable and carbon neutral energy sources. Algal lipid biosynthesis has received attention as a source for a carbon neutral biofuel. Equatorial and subtropical latitudes have been perceived as the most optimal regions for algal production. However, high evaporation rates in these regions have caused water resource issues in the industry. Our research focuses on the production of two marine algal species, Pavlova lutheri and Pavlova spp., in a mid-latitude setting with high seasonal variability in temperature and solar radiation. Two in situ incubation tanks (0.25 m³) equipped with HOBO temperature and total light monitoring systems were constructed and each filled with 50 L of F/2 algal growth medium made from seawater collected from Bellingham Bay. The tanks were deployed in Portage Bay during winter months when solar radiation and temperature were expected to be minimal (approximately 9.0 to 12.5 °C and 100 to 1000 µEm^-2s^-1 of photosynthetically active radiation). Cell counts and neutral lipid quantification (using BODIPY 505/515 dye) were measured every two days with a BD Accuri C6 flow cytometer. Analysis shows successful culture growth and lipid production occurs in these large outdoor incubation tanks. When cultures were grown in the labratory at 10 °C and 75 µEm^-2s^-1, maximum cell densities of 1.0 x 10⁷ cells/mL were achieved. With greater natural light abundance, we hypothesize that cell densities in our outdoor tanks will exceed those obtained under labratory conditions, and thus have higher oil yield per unit volume culture. 

Algal turfs are becoming increasingly dominant in subtidal marine environments, particularly in urban areas. Urchins have long been recognized as an important algal consumer on rocky temperate coasts, and can shift habitats from kelp forests to barrens. However, it is unclear whether urchins consume algal turfs. In a laboratory setting, I compared relative feeding and growth rates of juvenile green sea urchins, Strongylocentrotus droebachiensis, across four common algal types found in the subtidal in the Seattle Metropolitan Area. The algal types tested were the red foliose species, Chondracanthus exasperatus and Polyneura latissima, red algal turf (a combination of filamentous red algal species), and the brown alga, Saccharina latissimi. 120 juvenile urchins were collected from Mukilteo, WA and assigned randomly to one of these four treatments. Feeding rates were measured weekly and growth rates monthly, for a total of four months (Nov- Mar). I hypothesized that urchins would consume algal turf at the slowest rate, followed by red foliose macroalgae (C. exasperatus and P. latissima), and then kelp (S. latissima). In addition, I postulated that urchins feeding on algal turf would have a lower growth rate. Preliminary results suggested that urchin feeding differed significantly between algal treatments. Urchins consumed S. latissima at the highest rate followed by C. exasperatus, red algal turf, and P. latissima, respectively. Urchins that consumed S. latissima and C. exasperatus had a faster growth rate than urchins feeding on the other two algal types. These findings suggest that algal turf and P. latissima are consumed less rapidly than other algal resources. Algal turf and P. latissima are particularly dominant in Seattle’s urban marine environment, and their abundance may be one of the factors that limit urchin populations at early life stages locally.

What conditions are necessary for an ecosystem to function? All required elements must be present, but does their ratio, specifically of C and N, matter? Eutrophication has become a common problem characterized by excessive plant and algal growth due to the increased availability of one or more limiting growth factors needed for photosynthesis. However in natural water systems, CO₂ exchange is difficult to measure due to the constant exchange with the atmosphere; therefore we chose to study chemical balances by varying C:N ratios required to maintain both algae and Daphnia populations within closed ecological systems. Daphnia are planktonic crustaceans with bodies enclosed by an uncalcified shell (carapace) that belong to the Phyllopoda (sometimes called Branchiopoda), which are characterized by flattened leaf-like legs used to produce a water current for the filtering apparatus. In past experiments, algal cells removed too much CO₂ and HCO^3-. The remainder CO₃^2- caused a high pH to develop, and the Daphnia population did not persist. We hypothesized that a greater amount of NaHCO₃ per NaNO₃, would result in lower pH and allow longer Daphnia survival. Keeping the NaNO₃ concentration at 0.125 mM, we divided the 24 enclosed ecosystems into 4 treatment groups using base media with 3.3mM or 13.2mM NaHCO₃, and within each media type, 2 treatment groups of algae only and algae with Daphnia (6 replicates each). Our hypothesis predicted that the Daphnia population in the 13.2 mM NaHCO₃ media would survive longer. To date, day 28 of the experiment, 5 of the 6 replicates with the lower concentration, and all 6 of the 6 replicates of the higher NaHCO₃ have Daphnia populations. The experiment will be carried out longer to determine if the higher C:N ratio is more successful. This ratio could be used on preparing for any other closed ecological systems for longer periods of time.

Nature is full of examples of species that cooperate with one another to survive, but it is unclear how such associations can be stable in the presence of antagonists that may disrupt one or both species. The goal of this research is to explore the possibility that viruses disrupt the stability of an ecologically important mutualism between fermenting bacteria that release hydrogen and methanogens that consume the hydrogen and produce methane. Previously, 22 communities consisting of the bacteria Desulfovibrio vulgaris and the archaeaon Methanococcus maripauldis evolved for 1000 generations in the laboratory. At the onset of these mutualistic interactions, periods of instability leading to the extinction of two co-cultures were observed. One hypothesis is that this instability was caused by activation of viruses that had been dormant in the model bacterium. Here, we induce and isolate the viral pathogen from ancestral cultures, and intend to characterize the interactions that may lead to instabilities in these emerging mutualisms. Phage induction was achieved through mytomycin-C treatment of growing D. vulgaris cultures, and clarified with plaque assays and PCR. Phage were then applied to a susceptible host ssp. (Desulfovibrio vulgaris DP4) to innumerate and propagate the virus. Plaque assays and PCR indicated that extracellular phage endemic to D. vulgaris can be propagated in the susceptible DP4. We will use this phage to test instability in the experimental system, and to determine if bacteriophages have the capacity to cause extinction events in this tractable model system for evaluating microbial mutualisms.

The most common limiting factor for plant growth is nutrient availability. With revolutions such as the conversion of nitrogen gas into ammonia via the Haber-Bosch process, we have been able to sustain the agricultural growth our population demands. Endophytic bacteria provide an alternate solution to costly fertilizer production. Endophytes are bacteria that integrate into plant tissues and aid the plant in the recruitment of nutrients. The most well known endophytes are nitrogen-fixing Rhizobia, commonly found in the root nodules of legume plants. The various bacteria assayed in this project were found in wild poplar and isolated from various regions of the plant. Once isolated, the strains: Snoqualmie 117.2, WPR 5-2, WPL 5-2, WPL 7-2, WPL 8-1, WP 40, WP 41, and WP 42 individually promoted growth in poplar in comparison to non-inoculated controls. In this project, the 8 strains listed above were assayed for indole-3-acetic acid production (rooting hormone), insoluble phosphate solubilization, and siderophore production (iron chelation). The strains listed above were also assayed for possession of the nifH gene that is a subunit of nitrogenase, the enzyme that fixes atmospheric nitrogen. It is expected that not all of the bacteria within the given set will exhibit high levels of all of the assayed traits. However, the data will yield important information in understanding plant growth promoting factors within endophytic populations. The data can also be used to compare the quantified traits of isolated strains versus a consortium of multiple strains. Consortia of endophytes would be used for commercial or industrial purposes because of the potential efficiency of expressing high levels of a variety of plant growth promoting factors.

Previous research done in our lab found that poplar and willow trees grow in nutrient-poor soil and obtain their nutrients through partnership with microbes that live within them (referred to as endophytes). We have successfully isolated endophytes and are now exploiting them for plant growth promotion and stress tolerance. My research question is to test whether inoculating poplar clone OP-367 with endophytes can help the plant survive drought conditions. The experiment was set up by controlling watering schedule that simulates drought conditions and comparing the results of the uninoculated control group and the inoculated group. We quantified the results using measurements of wet and dry biomass, chlorophyll (SPAD readings) which quantifies the health of the plant by measuring the greenness, and maximal photochemical efficiency (Fv/Fm), which quantifies the photosynthesis happening in the plant. The implications of this project will be to permit better growth of plants under water stress conditions. The potential of endophytes to increase drought tolerance in OP-367 poplar hybrid and or other plant species can become a potential remedy for rescuing agriculture problems in relation to drought weather conditions.

Plant leaf cell growth is a function of the turgor pressure within the cells and the extensibility of the cell wall. The cell wall is a rigid physical barrier that prevents bursting from the osmotic pressure within the cell. In order for the cell to grow, this cell wall must be extended and the turgor pressure within the cell must be great enough to push the extension. The cell wall is loosened by acidification, caused by an efflux of protons across the plasma-membrane of the cell, allowing the extension. I have studied the common bean (Phaseolus vulgaris) to further the understanding of the functional relationship between turgor pressure, cell wall extensibility, and leaf cell growth and the effect of drought on these conditions. I grew my plants in the controlled environment of a growth chamber under well-watered and droughted (50% soil water content) conditions. Leaf growth rate was determined by daily measurements of the length of the leaf using a ruler. On the first trifoliate, I calculated turgor pressure using a pressure bomb and freezing point osmometer to measure water potential and solute potential, respectively. I found that a lower growth rate in droughted conditions is not met by a reduction in turgor pressure, which suggests that it is the result of reduced cell wall extensibility. Proton efflux or rate of acidification, measured using a pH probe in contact with the leaf cell wall, was reduced in the droughted plants, leading to a stiffer cell walls and resulting in the reduction of growth rate. I plan to continue my study by comparing multiple strains with variable drought tolerance. I predict that drought tolerance is associated with the ability to maintain the rate of acidification of the cell wall under droughted condition.

Stomata are pore-like structures on plant epidermis responsible for gas exchange, transpiration, and ultimately plant sustenance. Stomata require specific arrangements, such as proper spatial separation, in order to work correctly and efficiently. Studies have shown that cell-cell communication mediated by receptor-like kinases (RLKs) is critical for stomatal patterning. RLKs are transmembrane proteins that possess an extracellular receptor domain, which may bind signaling molecules and are thought to coordinate biological responses through their intracellular kinase domain. There are over 600 RLK genes in Arabidopsis, yet many of these genes remain unexplored. We hypothesized that RLKs specifically expressed during stomatal development may play roles in stomatal differentiation, polarity, and/or patterning. Previously, our group took advantage of genetic resources to enrich stomatal precursor cell states and performed transcriptomic analysis. Based on the transcriptome data, we selected three novel RLKs, tentatively named MV1, MV2, and MV3, that are highly expressed in stomatal precursors and sought to characterize potential functions of each gene. In order to investigate expression patterns of these genes, we generated transgenic plants expressing MV1, MV2, and MV3 under the control of their respective promoters carrying c-terminal yellow fluorescent protein (YFP) tags. Localization studies of third-generation YFP transgenic lines showed all genes to be expressed in epidermis of young developing tissue. Furthermore, MV1 is enriched in stem cell-like stomatal precursors while MV2 and MV3 appear enriched along dividing cell plates of stomatal precursor cells. Furthermore, we created lines expressing kinase domain ATP-binding site mutants, expecting the mutations to interfere with each kinase’s signaling networks and to incur dominant negative effects. However, observations of kinase-inactive mutants showed no phenotypic differences compared to wild type, possibly due to genetic redundancy. Therefore, we are pursuing kinase-deleted and knockout lines of each RLK and look to quantify any arising phenotypes.