Location: Plant Genetics Research
2024 Annual Report
Objectives
Objective 1: Conduct research to determine the impacts of genetic or environmental changes on central metabolism and flux in soybean to enhance productivity and seed composition traits.
Sub-objective 1A: Identify genotypes and phenotypes for further characterization.
Goal 1A: Identification of homozygous lines genetically altered to produce more protein and oil.
Sub-objective 1B: Use metabolite levels and isotope tracers to assess changes in carbon allocation that impact composition.
Goal 1B: In-depth analysis of molecular phenotype of altered seeds.
Objective 2: Develop and deploy novel analytical methods or tools to evaluate integration of various seed metabolism and turnover processes to produce desirable soybean seed composition and determine effects on the dynamics of seed metabolism.
Sub-objective 2A: Develop analytical tools to assess biosynthetic and turnover events in metabolism.
Goal 2A: Establish a method to quantify short chain acyl-CoAs.
Sub-objective 2B: Quantify intermediates of central and lipid metabolism that contribute changes in composition.
Goal 2B: Quantify lipid dynamics in seeds.
Objective 3: Conduct research to develop methods for identification and characterization of novel genetic variation for seed composition traits in soybeans using sequencing and/or genome editing to validate the genes and/or associated biological networks, and work with breeders to determine phenotypic variation and usefulness for improving soybean seed traits and climate resilience.
Sub-objective 3A: Identify genes, gene variants and networks important to soybean seed quality, yield, and resilience to climate change.
Goal 3A: Identify putative causative genes, alleles and associated regulatory gene network underlying QTLs important to soybean seed quality, yield, and resilience to climate change.
Sub-objective 3B: Validate and characterize the discovered genes, gene variants and networks to develop new strategies for biotechnologists and breeders to effectively improve soybean seed traits and climate resilience.
Goal 3B: Developing a manageable number of validated genes, gene variants and networks for effectively improving soybean seed quality and climate resilience.
Approach
Goal 1A: Identification of homozygous lines genetically altered to produce more protein and oil.
Soybeans will be modified through transgenic approaches to enhance seed value by augmenting metabolic pathways to alter composition in protein, oil, and carbohydrate. Transformed soybeans containing genes to reduce lipid turnover and to reduce carbohydrate biosynthesis will be self-pollinated, screened, and tested for oil and protein levels, seed size, pod and node number to choose transgenic events for further evaluation.
Goal 1.B: In-depth analysis of molecular phenotype of altered seeds.
Lipid, protein, and carbohydrate production will be quantified including new methods to assess synthesis of carbohydrate polymers involving development of combined mass spectrometry and UV-visible light spectrophotometer-based assays. Central intermediates, proteins, and lipids in combination with their isotopologues from isotope-labeling experiments will be quantified to derive metabolic flux information.
Goal 2.A: Establish a method to quantify short chain acyl-CoAs.
A method to extract and quantify short chain acyl-CoAs will be established through development of extraction and purification methods along with detection and quantification using liquid chromatography mass spectrometry. Odd chain length standards will be used with multiple types of column chromatography and injected into samples to assess extraction efficiency, serve as controls, and internal standards.
Goal 2.B: Quantify lipid dynamics in seeds.
Methods with isotopes including 13C, 18O, or 2H will be tested to develop a quantitative assay to assess the rate of fatty acid and lipid biosynthesis and turnover in filling seeds. To assess the rate of fatty acid biosynthesis, isotopically labeled acyl-acyl carrier proteins (i.e., acyl-ACPs) will be quantified by liquid chromatography mass spectrometry, and acyl-CoAs quantified to assess fatty acid and lipid synthesis and turnover.
Goal 3.A: Discover a suite of putative causative genes, alleles and associated regulatory gene network underlying QTLs important to soybean seed quality, yield, and resilience to climate change.
A bioinformatic pipeline will be developed to analyze the large soybean structural and functional genomic, genotyping and phenotyping, and other biological datasets. Integrative and data-driven strategies will be developed to mine -omics data for predicting and cross-validating causative genes, alleles and associated regulatory gene networks underlying QTLs important to soybean seed quality, yield and resilience to climate changes.
Goal 3.B: Validate two genes underlying two major QTLs for soybean oil, their gene variants and networks to effectively improve soybean seed quality and climate resilience.
Two major QTLs for seed oil and protein will be selected. Soybean plants over-expressing and containing altered gene functions will be generated and characterized to validate the underlying genes, alleles and gene regulatory networks and to comprehend resilience to environmental stresses. Transcriptomic and metabolomic changes will be investigated to illustrate their molecular mode of action.
Progress Report
Objective 1: Research included the generation of soybeans with altered carbohydrate biosynthetic pathways. Soybeans are economically valued for oil and protein in the seed, but also contain a significant amount of carbohydrates. Transgenic constructs to reduce the biosynthesis of carbohydrates were generated, soybeans transformed, and screened for homozygous lines. Assessing if reductions in carbohydrate are detrimental to the seed and whether they can result in increased oil levels is important to produce higher valued soybeans.
Objective 3: Whole genome sequencing data of over 12,000 soybean accessions and about 8,000 ribonucleic acids, (RNA) transcriptome sequencing data representing 2,800 biological treatments in the public domain and generated in the ARS laboratory were consolidated, quality controlled, and annotated. The deoxyribonucleic acid (DNA) and RNA sequencing data were analyzed to identify DNA sequence variants in 5,000 soybean accessions and gene expression of 56,000 soybean genes in the 2,800 biological treatments for further genetic and structural and functional genomic analysis to discover genes and networks important for soybean seed composition improvement.
Accomplishments
1. Reduced lipid breakdown results in soybeans with more oil and increased value. The value of soybeans is established by the amount of oil and protein in seeds. Oils are important for biofuels and as vegetable oil products used in cooking. ARS scientists in Columbia, Missouri, observed a drop in oil levels in soybean seeds over plant development and hypothesized the reduction could be due to lipid breakdown during seed maturation. Engineering to reduce the breakdown resulted in seeds with an increase in lipid and unexpectedly, bigger seeds. The bigger seeds were not the consequence of producing fewer seeds per plant, thus the oil produced per plant was significantly higher. Further measurements indicated that hormone levels were altered and may partially explain the observed increase in seed size. Producing bigger seeds that contain more oil per acre will result in increasing the value of soybeans which could be of significant benefit to farmers and agribusiness.
2. Development of a software tool to model lipid metabolism with radiolabeling data. Plant cells use biochemical pathways to produce the protein and oil that establish seed value. Isotopes are used to track the activity through the pathways but result in complex descriptions that can be laborious and challenging for scientists to analyze. ARS scientists in Columbia, Missouri, developed a software tool to model lipid metabolism using radiolabeling data. By understanding the flow of metabolites through different biochemical pathways, scientists can make rational engineering decisions to modify soybeans and other crops for improved yields and enhanced resilience to changes in the environment. Enhancing yield and tailoring seed compositions is crucial to meet demands for edible oils and to supply renewable, sustainable alternatives to petroleum, that add to farmer profits and support the U.S. economy.
3. Reduced anti-nutritional seed properties in soybeans resulting in improved quality for food and animal feed uses. Soybean is a major seed crop in the world and contributes $125 billion to the U.S. economy each year. Sixty percent of soybean value is from soybean meal for human food and animal feed. Unfortunately, soybean seeds contain a significant amount of trypsin inhibitors (TI), major anti-nutritional proteins for human food and animal feed. A toasting process is necessary for deactivating the trypsin inhibitors, which accounts for 25% of soybean processing costs. In collaboration with ARS scientists in Aberdeen, Idaho, ARS scientists in St. Louis, Missouri, applied gene editing technologies to inactivate a group of genes and develop new soybean plants containing more than 80% reduction of trypsin inhibitory activity. The modified soybeans can significantly increase soybean nutritional value and reduce processing costs, improving U.S. soybean competitiveness in the world market and with other seeds, and profiting U.S. farmers.
Review Publications
Gomez, J.D., Wall, M.L., Rahim, M., Kambhampati, S., Evans, B.S., Allen, D.K., Antoniewicz, M.R., Young, J. 2023. Program for integration and rapid analysis of mass isotopomer distributions (PIRAMID). Bioinformatics. 39(11). Article btad661. https://doi.org/10.1093/bioinformatics/btad661.
Mohanasundaram, B., Koley, S., Allen, D.K., Pandey, S. 2024. Physcomitrium patens response to elevated CO2 is flexible and determined by an interaction between sugar and nitrogen availability. New Phytologist. 241(3):1222-1235. https://doi.org/10.1111/nph.19348.
Kambhampati, S., Hubbard, A.H., Koley, S., Gomez, J.D., Marsolais, F., Evans, B.S., Young, J.D., Allen, D.K. 2024. SIMPEL: using stable isotopes to elucidate dynamics of context specific metabolism. Communications Biology. 7. Article 172. https://doi.org/10.1038/s42003-024-05844-z.
Xu, Y., Kambhampati, S., Morley, S.A., Cook, R., Froehlich, J., Allen, D.K., Benning, C. 2023. Arabidopsis acyl carrier protein4 and rhomboid like10 act independently in chloroplast phosphatidate synthesis. Plant Physiology. 193(4): 2661-2676. https://doi.org/10.1093/plphys/kiad483.