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INVESTIGATOR: Sanjaya, D.

PERFORMING INSTITUTION:
WEST VIRGINIA STATE UNIVERSITY
PO BOX 1000
INSTITUTE, WEST VIRGINIA 25112

GENETIC IMPROVEMENT OF SEED STORAGE COMPOUNDS IN OILSEED PLANTS

NON-TECHNICAL SUMMARY: Given the economic value associated with seed compounds such as oils, protein and starch, agriculture-breeding research has focused extensively on improving traits associated with these compounds. Metabolic engineering strategies have been instrumental in developing mechanisms to improve seed compounds in crops. Enhancing the genetic control of seed development and storage compound metabolism will lead to qualitative and quantitative improvement of oilseed crops. In collaboration with researchers at the University of Nebraska-Lincoln, researchers at West Virginia State University (WVSU) will use a combination of genomic, molecular biological and biochemical analyses to explore how molecular mechanisms regulate energy storage in oilseed crops, and how those mechanisms can be manipulated to increase energy storage--and thus the nutritional value--of the plants. WVSU undergraduate and graduate students will get hands-on experience in genome-editing, mutant screens, seed compound analysis by gas chromatography with flame ionization detector, and next-generation transcriptome sequencing. Knowledge gained from this research will unlock new and creative avenues for enhancing the molecular engineering of energy-dense oilseed crops. With increased concentrations of seed storage compounds, these crops will help to meet the growing food and fuel needs of the global population.

OBJECTIVES: Longer-term goals include identifying the mechanisms that regulate storage compounds in seeds (particularly oils, TAGs and proteins) in order to enhance the nutritive and energy capacity of oilseed plants. Our project has three research objectives: 1) investigate regulatory mechanisms controlling seed storage compounds in plants, 2) genetically enhance Camelina with storage compound biosynthesis genes, and 3) develop genome-editing system-based oilseed trait improvement.

APPROACH: The Sanjaya lab in collaboration with Co-PD labs, will explore the mechanisms that control storage compound synthesis and accumulation in oilseed plants. Developing seed transcriptome data of oilseed plants will be used for candidate gene identification. These candidate genes (at least 2-3) will be tested in Arabidopsis. The T-DNA mutant screening, overexpression/ RNAi lines and complementation analysis; analysis of number of seeds germinated, number of normal and defective seedlings, color of seedlings, morphology of root and hypocotyl and cotyledon/leaves, seed size, biomass; and storage compound analysis will be pursued to gain a mechanistic understanding of these candidate genes/enzymes. In addition, we will conduct post-sequence analysis, data interpretation, gene network analysis and qRT-PCR analysis for candidate genes. We will test gene-editing strategies in Camelina, including testing of enzymes and transcription factors involved in fatty acid and oil synthesis. We will alsodevelop diversified approaches to enhancing the application of PCR-based screening of TILLING populations and maintain homozygous lines in the greenhouse. Students will assist with data collection and analysis of plant height, leaf length, photosynthetic rate, stomatal conductance, transpiration rate, oilseed yield and metabolic data.

PROGRESS: 2020/04 TO 2021/03
Target Audience:Undergraduate and graduate students; plant biology researchers in academia, scientists in biotechnology industries; focus on agriculture, renewable energy, and post-coal mining reclamation companies. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project provided a training opportunity for undergraduate and graduate students during the reporting period. We incorporated active learning activities of plant transformation, transgenic crops, bioenergy, and environmental science, and concepts from the research under this project are into plant biotechnology class. How have the results been disseminated to communities of interest?Publication: S. Sanjaya, B. Muthan. (2021). Creating Power Foods with Gene Technology, Scientia, Issue 148, March. 3, pp 15-18 (In Press). Kiyoul Park, Sanju A. Sanjaya, and Edgar B. Cahoon Toward Sustainable Value-Added Industrial Oil Production in Plants. GCB-Bioenergy Bioproducts for a Sustainable Bioeconomy (under Review). M.S. Thesis: Deo B. (2020). Genetic study of abiotic stress tolerance and phytoremediation mechanism in the bioenergy crops camelina and switchgrass respond to coal mine soils. A Thesis Submitted to the Biotechnology Faculty of West Virginia State University in Partial Fulfillment of the Requirements for the Degree of Master of Science. Poster presentation: B. Muthan, Khathiwada, T. Heath, C. Challa, and S. Sanjaya. Increasing Energy Density in Switchgrass. Poster presentation in a symposium on Towards sustainable bioenergy production. Virginia Tech. February 2021. Oral Presentations: We presented some of this project results in WVSU green chemistry and first-year students' research experience classes. What do you plan to do during the next reporting period to accomplish the goals?Continue working on genetic, physiological evaluation of candidate genes function in Arabidopsis, evaluating genome editing plants and genotyping, screening Camelina homozygous transgenic plants, and molecular and biochemical analysis transgenic Arabidopsis and Camelina and yield evaluation.

IMPACT: 2020/04 TO 2021/03
What was accomplished under these goals? To identify the function of candidate genes in seed metabolism, we have checked the expression of these genes in Col-0 silique of 2, 4, 8, and 12-days after pollination using RT-qPCR. Interestingly, transcripts of these genes were significantly increased in siliques of 8 and 12-day after pollination, where most of the carbon allocated for oil biosynthesis, pointing to the connection of these genes in carbon partitioning and oil biosynthesis. However, few genes expression were high at the early stage of embryo development. That expression was declined at 12 DAP, suggesting that these groups of genes might have a role in embryo development. To validate these genes' connection to well-known seed storage compounds regulatory pathway regulators, such as the transcription factors WRI1, LEC1, and ABI3, we employed qRT-PCR analysis. Results show that candidate genesexpress during the seed filling stage, and they might have a role in fatty acid biosynthesis. To understand further, we have checked the expression in seed filling master regulators,wri1-1,lec1-11, andabi3-10, mutant's background. Most of thecandidate geneexpression was significantly up-regulated in thewri1-1mutant background but not inlec1-11andabi3-10, even though these regulators influence each and other expressions.The promoter::GUStransgenic lines of candidate genes show overlapped and distinct GUS expression patterns at an early and late stage of seedling establishment, respectively. Also, these lines show strong GUS expression in radicle at 1 and 2 DAS. We have generated multiple transgenic Arabidopsis plants expressing candidate genes/transcription factors that allowed us to increase storage compounds in seeds--without growth penalty and higher proteins, total fatty acids, TAGs, and calorific value. We have generated CRISPR/Cas9 genome-edited Arabidopsis plants. The growth of phenotypic variation was observed in a few transgenic events. Further validation at the genetic level is in progress. We have generated valuable metabolic data sets using transgenic plants expressing DGAT's alone or in combination with other genes. This data will allow us to determine the regulatory networks that drive storage compounds, which will support further studies in the design of nutrient-rich food and energy crops. Screening of homozygous Camelina lines expressing candidate genes alone or in combination and subsequent molecular and biochemical validation is in progress. We continually enhance WVSU's STEM undergraduate and graduate students' hands-on and classroom experience, helping them become skilled researchers and scientists in the STEM workforce.

PUBLICATIONS (not previously reported): 2020/04 TO 2021/03
1. Type: Journal Articles Status: Accepted Year Published: 2021 Citation: S. Sanjaya, B. Muthan. (2021). Creating Power Foods with Gene Technology, Scientia, Issue 148, March. 3, pp 15-18 (In Press).
2. Type: Journal Articles Status: Under Review Year Published: 2021 Citation: Kiyoul Park, Sanju A. Sanjaya, and Edgar B. Cahoon Toward Sustainable Value-Added Industrial Oil Production in Plants. GCB-Bioenergy Bioproducts for a Sustainable Bioeconomy (under Review).
3. Type: Theses/Dissertations Status: Accepted Year Published: 2020 Citation: Deo B. (2020). Genetic study of abiotic stress tolerance and phytoremediation mechanism in the bioenergy crops camelina and switchgrass respond to coal mine soils. A Thesis Submitted to the Biotechnology Faculty of West Virginia State University in Partial Fulfillment of the Requirements for the Degree of Master of Science.
4. Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: B. Muthan, Khathiwada, T. Heath, C. Challa, and S. Sanjaya. Increasing Energy Density in Switchgrass. Poster presentation in a symposium on Towards sustainable bioenergy production. Virginia Tech. February 2021.