Iowa State University, Iowa, USA 3. Research Fields

1. Name: Liza Esther ALEXANDER （ID No.: SP16001） 2. Current affiliation: Iowa State University, Iowa, USA 3. Research fields and specialties: Biological Sciences 4. Host institution: RIKEN- Center for Sustainable Resource, Kanagawa, Japan. 5. Host researcher: Dr. Kazuki SAITO (Group Director), Dr. Yozo OKAZAKI (Research scientist) 6. Description of your current research My PhD research is being conducted under the supervision of Dr. Basil J. Nikolau at Iowa State University (ISU), in the interdepartmental program of Molecular, Cellular and Developmental Biology. Plant epidermal cells express unique molecular machinery that juxtaposes the assembly of intracellular lipid components and the unique extracellular lipids that are unidirectionally secreted to the surface of the plant. The overarching objective of my project is to functionally characterize the genetic and metabolic networks that interrelate the intracellular and extracellular lipid metabolism. Physiologically this lipid-trafficking process is genetically programmed, but can change in response to environmental pressures (e.g., drought, temperature, pathogens), making them important to agricultural crop productivity. Additionally, these lipids are chemically most akin to petroleum hydrocarbons making this research insightful towards the development of biorenewable fuels and chemicals. My research builds on past research developments at ISU that has been and continues to be supported by the National Science Foundation (NSF) and the Department Of Energy (DOE). Specifically, ISU has developed maize systems for dissecting the genetic and metabolic networks and technological platforms for analyzing and imaging the metabolic intermediates of these processes. My research specifically focuses on the functional characterization of one of these maize gene family in lipid biogenesis. The strategy I am taking is two pronged: 1) expression of the protein encoded by the maize gene in a heterologous host that lacks this function; three such hosts are being used, bacteria E.coli, yeast Saccharomyces cerevisiae, and Arabidopsis mutant lines that carry mutations in a homologous gene; and 2) characterization of maize plants carrying mutations at the gene. In both strategies I have generated unique genetic stocks that are being analyzed by targeted and non-targeted metabolomics analytical platforms. Since the genes I am characterizing are known to affect extracellular cuticular lipid deposition I use GC-MS analysis to specifically profile these lipids and ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) to spatially image metabolites to individual cells, using well established procedures implemented by the Nikolau group at ISU. The proteins encoded by these genes express its biochemical function within the cell interior, specifically in the ER membranes possibly affecting fatty acid elongation. Therefore more sophisticated analyses are required to comprehensively explore the lipid profiles that can reveal the relationship between extracellular and intracellular lipid networks. This NSF EAPSI award in collaboration with the JSPS has provided valuable insights and helped me expand this area of my PhD thesis. 7. Research implementation and results under the program Title of your research plan: Evaluating intracellular and extracellular lipid metabolic networks at the level of individual cells Description of the research activities: The project focused on understanding the molecular genetics and metabolic relationships between the intracellular and extracellular cuticular lipid networks. The latter network is restricted to a single cell layer, the epidermis of plant aerial organs. Genetic stocks of maize and Arabidopsis designed at ISU that revealed changes in the extracellular lipid profile were used in this study, these include 1) transgenic Arabidopsis stems carrying specific maize genes 2) maize seedling leaves and 3) maize silks from inbred lines that showed differential expression in extracellular lipids. The comprehensive intracellular lipid profiles were analyzed via liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/Q-TOF/MS) guided by Dr. Yozo Okazaki, a Research Scientist in the Saito group. Integrating these new intracellular lipid data with the already generated extracellular lipid data provided invaluable insights on the interrelationship between the networks as disrupted by genetic permutations. The results from the transgenic Arabidopsis stems and maize seedling leaves showed that the specific maize genes used in the study influenced the intracellular levels of fatty acids of specific carbon chain lengths namely 30 and 32. The finding was able to support already generated extracellular lipid data that had shown changes 30 and 32 carbon length fatty acid derivatives. In addition to the changes in the fatty acid composition, several other lipid species were also seen to be influenced such as sphingolipids, glycolipids, acyl glycerols and polyprenols. Studies conducted at ISU by the Nikolau and Yandeau-Nelson groups have shown that the extracellular lipids are also richly expressed also in maize silks with a ~5-fold developmental increase in accumulation of these lipids along the length of the emerging silk. The intracellular profile of these maize silks also showed differences mainly between the different genotypes but with fewer differences across the developmental gradient of the silk. Several uncharacterized metabolites were also seen to be altered in the study and using tools developed at RIKEN – namely MS-DIAL and MS-FINDER, some of these metabolites have fully annotated and others partially identified. Although a lot more work needs to be done to understand the gene functionality, this project has demonstrated the utility of integrating molecular genetic strategies with advanced instrumentation for biochemical analyses (i.e., PTV-GCMS, LC/Q-TOF/MS and MALDI-FTICR imaging), and thus generated new knowledge concerning metabolic networks that occur uniquely within specialized single cell tissues. A manuscript highlighting these findings is in progress. 8. Please add your comments, including any cultural experience during your stay in Japan (if any): Amazing experience at RIKEN and Japan as a whole. The work ethics, hospitality and friendliness of those at RIKEN and around is something I would treasure forever. The beauty of Japan lies in its terrains, food, and mostly its people. Festivities celebrated with fireworks and dances, temple architectures, visits to Hakone and other surrounding areas along with the exquisite variety of food at every spot have definitely made a mark! 9. Advisor’s remarks (if any): I, Kazuki Saito hosting Liza Esther Alexander, must say that she made a magnificent job together with my colleague, Yozo Okazaki, regarding the detailed lipid analysis of her samples. She has been excellently integrated with our people in RIKEN for the last several weeks. I am quite convinced she will be able to pave the way to success for her scientific career in future. RESEARCH REPORT 1. Name: Matthew Barcus （ID No.: SP16002 ） 2. Current affiliation: Cornell University 3. Research fields and specialties: Biological Sciences 4. Host institution: University of Tokyo 5. Host researcher: Prof. Shinya FUSHINOBU 6. Description of your current research Chicken feathers are a large byproduct from the poultry industry, yet the high protein content of the feathers (>85%) offer a unique opportunity to be used as an animal feed and fertilizer, provided they are properly processed. Using enzymes to break down feathers has the potential to reduce energy requirements during the rendering process and yield a better end product. Wax esters are present on the surface of the feathers due to the birds preening, which protects their plumage. By removing the waxy barrier created from preening, we would expect the feather to be more easily rendered into coproducts. Soil microorganisms associated with the ability to degrade feathers were screened for lipolytic enzymes. These enzymes were further tested for the ability to degrade wax esters. The top performing enzyme was selected to further study its catalytic mechanism. Due to low sequence similarity with known protein structures, it was necessary to begin crystallization work in order to gain insight on how the enzyme is able to hydrolyze the bulky, hydrophobic substrates. Knowledge gained from the study may also provide insight on how the enzyme can be optimized for chicken feather rendering and many other industrial processes that have waxy substrates. 7. Research implementation and results under the program Title of your research plan: Structure determination and molecular modeling of the catalytic mechanism of a wax-ester hydrolase isolated from a feather-degrading soil microorganism Description of the research activities: The main component to the research project was trying to crystalize the wax-ester hydrolase. The purified enzyme was subjected to hundreds of different conditions to find optimal parameters for crystals to grow. From over 400 conditions there were two hits. Adjustments were made with the concentrations of salt, buffer, and precipitant in an attempt to grow larger and better crystals. Some crystals were then tested using an in-house X-ray to determine if the crystals were indeed protein and to evaluate how well the crystals would diffract. A homology model of the wax-ester hydrolase was also constructed to provide some insight on the enzyme and serve

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