Location: Plant Genetics Research
2022 Annual Report
Objectives
Objective 1: Develop novel analytical methods to understand the dynamics that underpin lipid metabolism to guide metabolic engineering efforts for lipid production in seeds.
Objective 2: Assess central carbon metabolism in altered plant tissues and develop strategies that can be used to assess plant metabolic changes for improving agriculturally relevant seed composition traits or yield.
Objective 3: Develop and make available new approaches to evaluate gene functions in gene networks and verify these tools by examining previously identified gene networks in soybean.
Objective 4: Discover, characterize, and make available genes for industry-relevant protein and oil traits from new and existing genetic populations created through various methods, such as fast neutrons, conventional crossing, reverse genetics (TILLING), or mining exotic diversity contained in the USDA National Plant Germplasm System.
Approach
Goal 1.1: Quantify major acyl-Acyl Carrier Protein (ACP) species of fatty acid
biosynthesis in soybeans.
We will develop biochemical methods with mass spectrometry to rigorously quantify acyl-ACPs. Acyl ACPs connect central metabolism with lipid metabolism and will provide an indication of when acyl-ACP synthesis may be bottlenecking the production of lipids under different circumstances which will be further considered through isotopic labeling and measurement of labeled acyl-ACPs.
Goal 1.2: Quantify labeling in phospholipid and neutral lipid pools.
We will isotopically label seeds and investigate the labeling in phospholipid and neutral lipid intermediates that we hypothesize are most indicative of specific pathway use for lipid production and that can be informative to engineer increased lipid production in the future. The mass spectrometry methods will involve optimization with high resolution instruments.
Goal 2: Analyze labeling in organic and amino acid pools in developing soybeans.
We will build a platform to transiently label seeds with 13C over short durations (minutes to hours) to investigate the allocation of carbon during specific aspects of seed development. These stages of development contribute to the final composition and are therefore important in establishing the final composition. Methods to rigorously analyze important intermediates including amino acids and organic acids will include fragment evaluation with direct injection mass spectrometry and validation with standards prior to quantification of differences in seeds of different ages.
Goal 3: Demonstrate that expression QTL genetic mapping is an effective approach to evaluate regulatory functions of genes in a co-expression network.
A eQTL mapping analysis will be conducted with seed transcriptome sequencing and genome sequencing data of the wild and cultivated soybean genotypes to identify the trans-acting eQTL, reveal the relationship of candidate regulatory genes/alleles and their associated genes and evaluate each regulatory relationship (edge) to generate a consensus soybean seed gene regulatory network. A set of CRISPR/Cas9 genome editing vectors for a regulatory gene (hub) will be constructed to alter its regulatory function in “transgenic” soybean for validation of its regulatory functions in the network.
Goal 4: Establish that integration of structural and functional genomic analysis of genetic soybean diversity with QTL studies is an effective approach to discovering seed quality genes and alleles.
Big data analysis methodologies and data mining strategies will be developed to integrate QTL mapping data, transcriptome and genome sequencing data, soybean seed gene regulatory networks with seed storage reserves and metabolic pathways to identify putative genes/alleles that cause the variation in oil and/or protein content in soybean. We will sequence transcriptomes of soybean seeds containing different alleles of a putative gene to validate regulatory function and provide insight into regulation of oil and/or protein production in seeds.
Progress Report
Objective 1: Research continued on the refinement of analytical methods that enable analyses of fatty acid metabolism. Acyl-acyl carrier protein is a scaffold protein that is involved with fatty acid biosynthesis. Methods to purify and quantify the fatty acids attached to the acyl carrier protein were advanced by testing different chromatography systems. Such methods will be important for improving seed quality and in particular augmenting lipid production in soybeans to supplant fossil fuel needs.
Objective 2: Research continued on characterizing the changes in central and lipid metabolism over seed development by examining seeds with altered steps in central metabolism. Seeds were harvested at different stages in the developmental process and protein, lipid, amino acid content, acyl-acyl carrier protein, and seed size were measured. The development of soybeans with added value compositions that include more oil and protein will result from better quantitative understanding of seed metabolism.
Objective 3: Research continued on collecting/generating and analyzing soybean transcriptomes generated by our laboratory and available in the public. A total of 3,930 soybean transcriptome sequences were analyzed and are being used to identify genes and regulatory networks for oil and protein improvement. The transcriptomes of soybean seed varying in causative QTL alleles underlying protein and oil QTL were sequenced. The gene regulatory network underlying the QTLs is under construction and will be important to providing a road map to improve seeds genetically.
Objective 4: Research continued on analyzing whole genome sequences that we collected to identify DNA variants. Several algorithms were tested to identify DNA structural variants. A set of algorithms were developed to annotate DNA variation including single nucleotide polymorphism and structural variants. A variety of data-mining strategies are being tested to discover gene and causative variants underlying seed quality traits, which is important to develop new soybean cultivars through breeding and editing technologies.
Accomplishments
1. Demonstrated soybeans with altered enzymatic steps show differences in composition that can lead to renewable fuel alternatives and improved human diets. Soybeans are comprised of protein and oil that are valuable for food and fuel. Soybeans with changes in biochemical steps were analyzed through the growth of the plant and seed production to establish changing levels of nutrients in the seed. ARS researchers in Saint Louis, Missouri, showed that the oil level in some lines was increased with oil compositions that are healthier. Amino acids that are important for the human diet were also altered. The studies provide a strategy to improve seed composition with better oils and protein for human consumption and are important for efforts to produce renewable alternatives to petroleum-based fuels.
2. Developed new methods for measuring biochemical intermediates that indicate how soybeans accumulate lipid and protein that establish the seed value. Soybeans produce a significant amount of lipids that are valued as a source of edible vegetable oil and in biodiesel and renewable feed stock applications. ARS scientists in Saint Louis, Missouri, developed new methods to analyze the components that are required for production of lipids. The measurement of fatty acid precursors in soybean over the stages of soybean filling and maturation indicated that lipid production declines at mid-development and the total accumulated lipid in seeds is reduced by maturation. The results are important because the flow of carbon and nutrients through metabolism is challenging to quantify but dictates the final seed composition. Improving composition would have societal benefit by enhancing oil content and protein quality in soybean.
3. Developed a translational genomic resource to facilitate soybean research throughout the world. With the advance of high throughput technologies, scientists have generated a massive amount of biological data on soybeans providing an unprecedented opportunity for agricultural research. The biological data is currently under-utilized because few labs have the capacity to analyze it. The development of big- data technologies and resources can facilitate soybean research leading to discoveries and product development. ARS scientists in Saint Louis, Missouri consolidated, characterized and analyzed the genomes of 1,500 diverse publicly available soybean lines and documented approximately 32 million individual mutations in the genome. The comprehensive description was converted to a a user-friendly, searchable genomic library for others to easily access. The study also demonstrated their versatile uses in soybean genetics, breeding and molecular biology research. The genomic resource is available at SoyBase (https://soybase.org) and Ag Data Commons (https://data.nal.usda.gov/) for the research community.
4. Discovery of a key gene influencing seed protein, oil and yield and new strategies to improve soybeans. Seed protein, oil content and yield are highly correlated traits that account for the economic value of soybean. Seed protein is negatively correlated with seed oil and yield, which makes it challenging to increase levels of both. However, their underlying molecular mechanisms and their selection through breeding over the process of domestication is not understood. ARS scientists in Saint Louis, Missouri discovered a gene, named POWR1 (Protein, oil, weight regulator 1), which soybean researchers have been unable to clone over the past three decades. The POWR1 gene partially controls soybean seed protein, oil, weight and yield. A mutation in the POWR1 gene resulted in substantially increased seed oil content, weight, and yield and reduced protein. The study provides insight into how the POWR1 gene controls these economically important traits. Scientists also demonstrated that POWR1 also played a key role in soybean domestication and improvement. Evidence suggests that ancient farmers selected for larger seeds during domestication which led to increased seed yield/seed weight/oil but reduced protein content. The study identified a collection of soybean lines that are highly valuable for soybean breeders in the development of soybean cultivars containing improved seed protein. The work is significant as it will provide a strategy for researchers to improve soybean seed quality and yield, that can contribute to US soybean grower incomes.
Review Publications
Chu, K.L., Koley, S., Jenkins, L.M., Bailey, S.R., Kambhampati, S., Foley, K., Arp, J.J., Morley, S.A., Czymmek, K., Bates, P.D., Allen, D.K. 2022. Metabolic flux analysis of the non-transitory starch tradeoff for lipid production in mature tobacco leaves. Metabolic Engineering. 69:231-248. https://doi.org/10.1016/j.ymben.2021.12.003.
Aznar-Moreno, J.A., Mukherjee, T., Morley, S.A., Duressa, D., Kambhampati, S., Chu, K.L., Koley, S., Allen, D.K., Durrett, T.P. 2022. Suppression of SDP1 improves soybean seed composition by increasing oil and reducing undigestible oligosaccharides. Plant Biotechnology Journal. 13. Article 863254. https://doi.org/10.3389/fpls.2022.863254.
Koley, S., Chu, K.L., Gill, S.S., Allen, D.K. 2022. An efficient LC-MS method for isomer separation and detection of sugars, phosphorylated sugars, and organic acids. Journal of Experimental Botany. 73(9):2938–2952. https://doi.org/10.1093/jxb/erac062.
Wang, J., Kambhampati, S., Allen, D.K., Chen, L. 2022. Comparative metabolic analysis reveals a metabolic switch in mature, hydrated, and germinated pollen in arabidopsis thaliana. Frontiers in Plant Science. 13. Article 836665. https://doi.org/10.3389/fpls.2022.836665.
Wang, X., Liu, X., An, Y., Zhang, H., Meng, D., Jin, Y., Huo, H., Yu, L., Zhang, J. 2021. Identification of glutathione peroxidase gene family in ricinus communis and functional characterization of RcGPX4 in cold tolerance. Frontiers in Plant Science. 12. Article 707127. https://doi.org/10.3389/fpls.2021.707127.
Goettel, H.W., Zhang, H., Li, Y., Qiao, Z., Jiang, H., Hou, D., Song, Q., Pantalone, V., Song, B., Yu, D., An, Y. 2022. POWR1 is a domestication gene pleiotropically regulating seed quality and yield in soybean. Nature Communications. 13. Article 3051. https://doi.org/10.1038/s41467-022-30314-7.
Zhang, H., Jiang, H., Hu, Z., Song, Q., An, Y. 2022. Development of a versatile resource for post-genomic research through consolidating and characterizing 1500 diverse wild and cultivated soybean genomes. BMC Genomics. 23. Article 250. https://doi.org/10.1186/s12864-022-08326-w.
