Location: Plant Genetics Research2021 Annual Report
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.
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.
Objective 2: “Develop novel analytical methods to understand the dynamics that underpin lipid metabolism to guide metabolic engineering efforts for lipid production in seeds”. ARS scientists in St. Louis, Missouri performed experiments to evaluate isotopic signatures in lipids and their precursors. Seeds, such as soybeans have high value because they contain protein that is a primary component in animal feeds, and lipids that are a crucial source of edible vegetable oils, industrially important for biofuels and as a chemical feed stock. Understanding how plants allocate resources is important to efforts to produce seeds with increased value. Isotopes provide a means to assess active metabolism, therefore establishing methods to inspect tissue metabolism with isotope tracers is integral to the understanding of metabolism. To assess metabolism over stages of seed development, transient labeling methods were established in developing seeds. The methods resulted in labeled intermediates of central and lipid metabolism differentially with stage of development. The metabolic labeling signatures are integral to the production of lipids and protein and can be examined to understand the necessary changes to augment composition. Objective 3: “Assess central carbon metabolism in metabolically altered plant tissues and develop strategies that can be used to assess plant metabolic changes for improving agriculturally-relevant seed composition traits or yield”. Metabolites from wild type and transgenic soybeans at different stages in the seed development process were quantified and a subset cultured with isotopes. 13C substrates that are rapidly interconverted in central metabolism including glucose, sucrose, glutamine, glutamate, asparagine, acetate, and glycerol were pursued. The glycerol was particularly informative late in seed development. Lipids drop as a percent of the total seed composition late in development. The glycerol studies indicated lipid may be turned over for production of other less valued components in the seed at this stage of development when carbon is no longer being received from vegetative parts of the plant. Other studies were conducted to examine tradeoffs in amino acid and fatty acid levels as a result of changes to central metabolism in augmented lines. The combination of investigations explained observed changes in seed composition which contribute to seed value in soybean and are relevant to other important crop seeds.
1. Changes in soybean metabolism impact lipid production and distribution. Lipids in soybeans are important for biodiesel applications, vegetable oil production, and as a chemical renewable feed stock. ARS scientists in Saint Louis, Missouri, conducted studies with collaborators using isotope labeling, and imaging technologies combined with mass spectrometry to understand the metabolically active parts of seeds. The images resulted in spatial maps of lipid metabolism during development and indicated the ways in which seed composition changes as the seed develops. The observed changes likely contribute to differences in final seed oil and protein levels and are important to scientists interested in breeding or engineering seeds with altered composition.
2. Changes in soybean seed composition over late development partially diminish the seed value. The analysis of soybean at five temporal points in development established compositional changes within the seed due to alterations in metabolism. Soybeans are grown for protein and oil which establish the economic value of the seed; however, ARS scientists in Saint Louis, Missouri, showed that oil and protein are reduced at later stages of seed development. The lost protein and oil is replaced by insoluble carbohydrates and oligosaccharides and production of these carbohydrates also coincides with the disappearance of transitory starch in the seed. Since the most common oligosaccharides in soybean, raffinose and stachyose, cannot be digested by monogastric animals (i.e. animals with simple digestive systems) they are of lesser value to animals like pigs and chickens. The production of the oligosaccharides comes from an endogenous source of carbon because the scientists confirmed that the plant is no longer contributing nutrients for the seed late in development. Thus, the carbohydrates made late in metabolism are partly the result of turnover of other valued storage reserves like oil and protein. Taken together, this work indicated that one strategy to improve soybeans for use as animal feeds is to minimize the undesirable conversion of oil and protein to undigestible carbohydrates late in development.
Romsdahl, T.B., Kambhampati, S., Koley, S., Yadav, U.P., Alonso, A.P., Allen, D.K., Chapman, K.D. 2021. Analyzing mass spectrometry imaging data of 13C-labeled phospholipids in camelina sativa and thlaspi arvense (pennycress) embryos. Metabolites. 11(3). Article e148. https://doi.org/10.3390/metabo11030148.
Jenkins, L.M., Nam, J., Evans, B.S., Allen, D.K. 2021. Quantification of acyl-acyl carrier proteins for fatty acid synthesis using LC-MS/MS. Methods in Molecular Biology. 2295:219-247. https://doi.org/10.1007/978-1-0716-1362-7_13.
Aubuchon-Elder, T., Coneva, V., Goad, D.M., Jenkins, L.M., Yu, Y., Allen, D.K., Kellogg, E.A. 2020. Sterile spikelets contribute to yield in sorghum and related grasses. The Plant Cell. 32(11):3500-3518. https://doi.org/10.1105/tpc.20.00424.
Kambhampati, S., Aznar-Moreno, J.A., Bailey, S.R., Arp, J.J., Chu, K., Bilyeu, K.D., Durrett, T.P., Allen, D.K. 2021. Temporal changes in metabolism late in seed development affect biomass composition. Plant Physiology. 186(2):874-890. https://doi.org/10.1093/plphys/kiab116.