This study focuses on the relationship between two OMP85 homologs, Toc75 and OEP80, derived from an ancestral cyanobacterial protein. These two homologs are present within modern-day chloroplasts. OEP80 is hypothesized to play an important role in seedling development much like its sister protein – Toc75. This study attempts to determine the function of OEP80 as well as its necessary expression time (in either the seed, germination, or seedling stage). By creating mutants without a properly functioning OEP80 gene and rescuing these mutants using genetic constructs, this study compares the functional sequences of OEP80 and Toc75. These constructs can then be used to determine the exact time (stage of plant development) and role that the OEP80 plays in functioning and normal development of plants.
Tyrosine sulfation is a post-translational modification of proteins that is important to protein-protein interactions. We show that integrins, a family of trans-membrane receptors found in animals that link cells to the Extracellular Matrix, have a high number of likely tyrosine sulfation sites. Particularly, likely tyrosine sites in integrins are often found near ligand binding or recognition sites. Our data also show a high degree of conservation between likely sulfated tyrosine sites, suggesting their functional importance to the integrin.
Nitrogen is a nutrient essential to plant growth and development. Improving a plant's ability to uptake and metabolize nitrogen is essential to preserving plant productivity and agricultural viability under low nitrogen growing conditions, but is difficult to achieve due to the complicated nature of the nitrogen assimilation pathway. Because transcription factors often regulates the coordinated expression of genes involved in a pathway, modification of transcription factors is a powerful and promising approach towards modifying complicated pathways. This experiment focuses on the effects that the Dof1 transcription factor has on nitrogen assimilation in tomato plants in hopes to design a tomato that can maintain high crop yield without requiring added nitrogen from fertilizers. Plant length, fresh weight, dry weight, root length, and PEP-Carboxylase expression were higher under low nitrogen conditions in plants overexpressing Dof1, showing that the Dof1's role in regulating nitrogen metabolism in plants has a positive impact on nitrogen assimilation in transgenic tomato plants under low nitrogen conditions. Although this project evaluates the NUE of a transgenic tomato plant, the ultimate goal is to create a non-transgenic line that has similar properties.
Antiporters are generally responsible for ion homeostasis in the model organism Arabidopsis thaliana by exchanging hydrogen ions (H+) for sodium (Na+) or potassium (K+). However, information regarding the role of NHX2 and NHX3 in this process is scarce; therefore, this study aims to investigate the effect of overexpression of NHX and NHX3 genes individually when the plant is grown in Spalding media with varying concentrations of potassium: 30mM (high), 1mM (medium), and 0.1mM (low). After ten days of growth, root length was measured and then expression of HAK5 and AKT1, which correlate with low levels and high levels of potassium, respectively, were measured using comparative qPCR analysis. The results indicated that though overexpression of the NHX2 gene had little to no effect in any concentration of potassium media, overexpression of the NHX3 gene significantly impacted growth and development. Specimens with an overexpressed NHX3 gene exhibited shorter roots, increased expression of HAK5, and decreased expression of AKT1 in low levels of potassium. Not only do these results present a better understanding of Arabidopsis thaliana, but they may also guide the process of developing better stress response capabilities in crops; abiotic stress is the primary cause of crop loss worldwide.
Medusahead (Mh, Taeniatherum caput-medusae, (Sim). Nevski, Elymus caput-medusae), a noxious, invasive, annual grass, has invaded millions of acres in west coast states and is destructive to the natural ecosystem. Mh invasions decrease biodiversity, commercial and wildlife grazing value, and the capacity and recreation value of rangeland. Mh has a maximum period of susceptibility in the spring to control methods, but the start and end point are unknown. Thus, control methods, such as mowing, grazing, fire and herbicides, are ineffective or not feasible. The purpose of this project is to find the end point in that period to improve the timing of controls. First, a classification of partial Mh phenology, from post-pollination to complete maturity, was constructed. Then, seeds of different life stages from three different regions in California were germinated to find the germination percentages of each stage. The data, along with the results of a 1957 seed germination experiment, was then analyzed to identify the end point and to examine regional differences in Mh viability. The latest phenological stage at which Mh is still susceptible to control was found to be Intermediate 2. Also, differences in germination among the regions were identified.
Tyrosine sulfation is a posttranslational modification of a protein in which the hydroxyl group of the amino acid tyrosine is changed into a sulfate group. This modification strengthens protein-protein interaction. Tyrosine sulfation is prominent in the signature sequence of the conserved selectivity filter of voltage-gated potassium channels. The functionally similar KcsA from bacteria Streptomyces lividans shares this signature sequence. Positively charged toxin binding sites interact with negatively charged sulfated tyrosine sites. Voltage-gated potassium channels function in repolarization of action potentials in the brain, heart, and muscles. Tyrosine sulfation plays a critical role in the conduction of these channels.
Standard microbial culture-based methods were used to study the composition and diversity of three vermicompost samples (Terra Vesco, Sonoma, CA, USA). Bacterial colonies were isolated from vermicompost using serial dilutions. The amplification and sequence analysis of 16S rDNA from 39 isolates showed a high percentage (79%) of Actinobacteria sp., especially Arthrobacter sp. (54%) and Streptomyces sp. (15%). Proteobacteria and Firmicutes sp. were also detected. The level of bacteria diversity in the soil supported the idea that microorganisms in vermicompost may be able to outcompete and reduce soil-borne pathogen populations, making vermicompost a viable disease-control method for the agriculture industry.
Perennial crop plants like grapevine (Vitis vinifera L.) use stored carbohydrates (CHO) from the previous season to sustain early season growth and flower development. Water deficits inhibit current season growth and yield of the current season and following season, but the effects on stored CHO are not known. CHO assays were performed on two grapevine genotypes, Grenache and Syrah, across three different irrigation treatments. Specifically, CHO concentrations in basal nodes and internodes were assayed just after the onset of veraison, the onset of ripening, which has been shown to be the annual low point in storage CHO concentrations in grapevine. A series of hot ethanol baths were used to extract soluble sugars. After extraction, CHO samples were digested into glucose using an alpha-amylase and amyloglucosidase solution. A PGO enzyme o-dianisidine dihydrochloride solution was added to each sample for a color reaction. The plate was read using a spectrometer and optical density numbers were recorded and analyzed with a standard starch calibration curve. In general, Syrah had higher storage CHO concentration than Grenache, which suggested that it was more efficient with water. Across both varieties and all three irrigation treatments, internodes contained higher concentration of storage CHO. CHO distribution in internodes supported the hypothesized relationship: decreased water availability results in decreased concentrations of storage CHOs. These data suggest that water deficit and storage CHO concentrations are positively correlated in perennial crop plants like grapevine (Vitis vinifera L.). CHO in the nodes did not show the predicted distribution. Syrah vines had lower midday leaf water potential than Grenache vines which can be attributed to their anisohydric tendencies. This information on CHO distribution and genotypic variation in response to water deficits is important for identifying water-efficient grapevines and for water-conservation in agriculture.
Panelists were given blind samples of body washes to evaluate at home on a seven point scale. They received sufficient training to be able to distinguish between product differences for each attribute of the body washes. The goal of this study was to improve on the current methods of analytical product assessment with a home use descriptive analysis study to reflect more realistic home use situations of body wash products.
Evolution of 2-Nitrotoluene 2,3-dioxygenase and Gene Manipulation of Catechol 2,3-dioxygenase in Acidovorax sp. Strain JS42
Acidovorax (formerly Pseudomonas) sp. JS42 is able to use the compounds 2-nitrotoluene (2NT) and nitrobenzene as carbon, nitrogen, and energy sources. In the first step of the degradation pathway, 2-nitrotoluene 2,3-dioxygenase (2NTDO) converts 2NT to 3-methylcatechol with concomitant nitrite release. 2NTDO has three component proteins, and although it can break down all three mononitrotoluene isomers, JS42 cannot grow on 3-nitrotoluene (3NT) or 4-nitrotoluene (4NT). However, it is possible to force JS42 to adapt to 3NT and 4NT with resulting changes to 2NTDO. In this study, we evolved 4NT+ JS42 strains to grow on 3NT using long-term selection. Sequencing of the ntdAc gene encoding the α subunit of 2NTDO oxygenase revealed substitutions at the amino acid position 204 on the active site. Mutations at positions 238 and 248 were conserved from the parent 4NT+ strain and no other mutations were present, supporting the hypothesis that the amino acid position 204 is crucial for the evolution of JS42 to utilize new substrates. The next step of the 2NT degradation pathway involves catechol 2,3-dioxygenase (CDO). Both ctdE1 and ctdE2 encode CDO, and each has an associated regulatory gene (ctdR1 and ctdR2). Based on the phenotype of a mutant strain with deactivated ctdE1, ctdE1 seems critical to the degradation pathway. We will complement the ctdE1 mutants to verify the role of the enzyme in 2NT degradation. It is not yet known whether ctdR1 encodes an inducer or repressor, so ctdR1 inactivation will be used to determine the function of the regulatory protein.
According to the Institutional Animal Care and Use Committee (IACUC), Public Health Services (PHS), and Office of Laboratory Animal Welfare (OLAW), institutions must follow guidelines set by the Guide for the Care and Use of Laboratory Animals in their animal care and use programs when establishing mice populations within a cage to maintain the health and welfare of the laboratory mice2. The effects of mouse-caging density were studied in this project to determine the maximum number of mice that could be housed in a cage and maximum time between cage changes while maintaining the health and welfare of the mice. The CO2 level, Ammonia level, change in air flow, temperature, humidity, and any social, behavioral, or physical changes of the mice were evaluated. Seventy cluster of differentiation 1 (CD-1) outbred female adult mice were divided into four groups, with a minimum of two mice and a maximum of five mice per cage. Each week measurements of the microenvironmental gases in the cage, observations of the behavior and physical changes in the mice, cage temperature, cage humidity, cage airflow, and weights of the mice were recorded, for three weeks.
The atmospheric carbon sequestration abilities of plants, and in specific of grasses, are important on a local and global scale. However, little is known about the difference in carbon sequestration rates of perennial and annual grasses. The goal of this study is to discover if and to what extent grassland restoration, which is commonly achieved by seeding native perennial grasses, increases carbon sequestration through the analysis of soil organic carbon (SOC) stocks. Two sets of soil samples were taken, one set with samples from restored and nearby unrestored sites, and one with pairs of adjacent annual and perennial samples. The soils were processed and analyzed for carbon content in external laboratories. Statistical analysis using JMP revealed that the unrestored sites generally have higher amounts of SOC, although there is a positive relationship between perennial grasses and carbon presence. We conclude that the varying management practices and land use histories greatly shape SOC stocks in grasslands and that there is preliminary evidence that perennial grasses are more effective than annuals in storing carbon.
Milk oligosaccharides play a prebiotic role in the growth and establishment of a balanced gut flora in infants, selectively enriching the beneficial and protective bacteria that promote healthy immune and cognitive development. By acting as anti-pathogenic agents, milk oligosaccharides hold great promise as high quality supplements that can help support the health of any population with comprised immune systems. In order to provide sufficient quantities of oligosaccharides for clinical and functional testing, this work optimized several steps of activated carbon oligosaccharide filtration from human milk permeates. For measurement of process efficiency, a carbohydrate assay was first developed by treating milk products treated with Carrez clarification. Carbohydrate adsorption tests on activated carbon followed by total carbohydrate and lactose quantification showed that ratios of activated carbon to carbohydrate from 5-10 had better carbohydrate retention. In addition, permeates filtered with finer mesh charcoal (20-500) at a charcoal/carbohydrate ratio of 10 had the majority of carbohydrates adsorbed. Optimized conditions found in this study can be applied to purification from bovine milk whey permeate, a dairy industry waste stream. With further refinements to other steps of the purification technique, activated carbon filtration can be adapted as an effective method for mass extraction of health promoting oligosaccharides.
Uridine 5’diphopho-α-D-galactopyranoside (UDP-Gal) is a sugar nucleotide commonly used by animals, plants, and bacteria as the activated donor of galactose (Gal). The salvage pathway is the simplest route to synthesize UDP-Gal and involves three different enzymes: Streptococcus pneumoniae TIGR4 galactokinase (SpGalK), Bifidobacterium longum UDP-sugar pyrophosphorylase (BLUSP), and Pasteurella multocida inorganic pyrophosphatase (PmPpA)1. The optimum conditions for the entire reaction pathway in a one-pot multienzyme system were determined. Then, using these conditions, synthesis of UDP-Gal was scaled-up to the gram-scale. Production of UDP-Gal was successfully attempted using 1 g of Gal and a reaction yield of 70% was obtained. No isolation yield was determined because of incomplete purification.
Shade avoidance is a series of plant responses elicited when a plant experiences a reduced ratio of red to far-red (R:FR) light. In this study we investigate the genetic basis of shade avoidance by examining hypocotyl and root growth of Arabidopsis thaliana in four natural variants and three mutant lines. The four natural variants were Columbia (Col), Kondara (KON), KNO-18, and RRS-10. The three mutant lines, numbered 19, 38, and 52, were TDNA lines with insertions in the gene of interest. The average hypocotyl length was found to be longer in the shade conditions for every genotype. From the difference between the average hypocotyl length in sun and shade conditions, it was found that 19 may be a high responder, 38 responds similarly to wild-type Col, and 52 may be a low responder. After 3 days of shade treatment, there was little difference between the average root length between genotypes or between plants grown in sun or shade conditions. However, primary root length measured after 10 days of light treatment revealed that the average root length was longer in the shade conditions for every genotype. This suggests that roots show increased growth in response to shade. These results are relevant because knowledge gained from this experiment can be utilized in the agricultural industry when deciding crop spacing and land conservation.