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United States Department of Agriculture

Agricultural Research Service

Research Project: GENETIC ENHANCEMENT OF SOYBEAN SEED VALUE BY BIOTECHNOLOGY

Location: Plant Genetics Research

2012 Annual Report


1a.Objectives (from AD-416):
1) To develop soybeans with altered seed coat color to facilitate the segregation and identify preservation of seeds with genetically enhanced compositional traits..
2)To produce soybeans with enhanced food, feed, and industrial properties by modification of the oil and lipid-soluble antioxidant composition of seeds..
3)To introduce genes into soybeans that result in high-level accumulation of foreign and engineered proteins valuable for food, feed, and industrial uses..
4)To develop procedures and methods for analyzing and predicting seed protein allergenicity in food and feed. To develop non-allergenic seeds by supressing intrinsic allergens and to modify proteins that are potential transgenes for improving biosafety.


1b.Approach (from AD-416):
1) Co-transformation of soybean with a seed coat color-conferring transgene and a trait transgene will yield visually distinct seeds with enhanced composition..
2)Identify enzymes that are specialized for the metabolism of unusual fatty acids to produce agronomically-viable soybean seeds..
3)Manipulate protein content of soybean seeds by use of plant promotors and compartmentalization of introduced proteins..
4)Identify and characterize soybean food and feed allergens.


3.Progress Report:
We screened the elemental composition of >30,000 soybean seeds including macronutrients in fertilizer, elements of significance to plant and human health and elements causing agricultural or environmental problems. This system was used to profile soybean seed composition as a function of canopy position within experiments altering the environment. Several elements showed significant differences through the canopy for all of the cultivars and environments, while others were specific for given environments and cultivars. Understanding what causes these differences will give a better understanding of adaptations the plants are making to the environment.

Genes important for elemental accumulation were identified by profiling 1500 diverse cultivars. Lines showing extremely high or low levels of Sulfur (S), Phosphate (P), Cadmium (Cd), Manganese (Mn) and Aluminum (Al) have been sent to researchers and breeders studying seed S and P, the response to high Cd and Al soils and Mn deficiency soils respectively. We confirmed 16 seed elemental composition mutants identified from a genetic screen to be used to identify the genes under field-grown conditions. The results were used to design a screening strategy that will require 50% fewer samples to perform. We profiled a mapping population derived from soybean cultivars that differ in iron availability and identified several genetic loci affecting seed composition. This will allow identification of important genes for improving yield with less fertilizer or on low quality soils.

To explore the metabolism of developing soybean seeds scientists in St Louis, MO previously developed an embryo culturing method which has been used to quantify oil, protein, and carbohydrate levels in seeds that were exposed to different concentrations of sugars and amino acids recorded as a ratio of carbon to nitrogen. These environmental perturbations caused drastic changes in protein levels and altered the fluxes through primary metabolism.

These investigators fed a subset of cultures with 13C-isotopes (i.e. amino acids and sugars) and developed methods to assess the label incorporation as a result of metabolism. Radiolabeling experiments were used to monitor differences in carbon allocation. Flux maps were built to better understand the changes in metabolism that can guide rational metabolic engineering efforts.

Metabolic events take place dynamically (in time) and occur in distinct locations (cellular and subcellular compartmentation) and limit computational systems biology approaches. Scientists in St. Louis, MO labeled developing seeds with 13C isotopes provided as sugars and amino acids. They used this biomass to develop a method to resolve the labeling in different proteins with mass spectrometry. Current investigations focus on developing improved methods that may allow examination of protein turnover and other temporal events. Together, these investigations provide advanced levels of detail on metabolism that are critical to understanding plant biology and function at molecular levels.


4.Accomplishments
1. Identified genetic factors affecting maize kernels. Human health and the efficiency and sustainability of agriculture all depend on the ability of crop plants to acquire and use mineral nutrient resources. The suite of mineral nutrients found in a crop plant are called the ionome, and the study of their interactions is called ionomics. ARS scientists in St. Louis, MO and Ithaca, NY described the ionome of maize grain samples grown on fields in Florida, New York and North Carolina. They described correlations within and between the datasets, finding several genetic factors that helped the maize plant acquire and store mineral nutrients such as calcium, potassium, and phosphorous on all of the sites. This is in spite of the dramatic differences in soil and climate between the three farm sites. This represents a starting position for improving the mineral nutrient quality of maize, to improve human health and enhance agricultural productivity.

2. Sampled the diversity of elemental accumulation in a model plant. Understanding how plants regulate element composition of tissues is critical for agriculture, the environment, and human health. Sustainably meeting the increasing food and biofuel demands of the planet will require growing crops with fewer inputs such as the primary macronutrients phosphorus (P) and potassium (K). Ionomics is the study of elemental accumulation in living systems using high-throughput elemental profiling. With this technique, ARS scientists in St. Louis, MO can rapidly generate large quantities of data on thousands of samples, allowing for the identification of genes important for elemental accumulation. They used this approach to sample the natural diversity present in collections of a model plant, the wild mustard Arabidopsis. They found that the elemental composition of the plant is tightly controlled by its genes. They also found that elements will have different relationships between them depending on the environment and the tissues (root, seed or leaf) under study. This suggests that crop varieties developed for improved elemental uptake and accumulation will be highly environment specific. These findings contribute to a novel strategy to improve the productivity of all major crops thus impacting world food security in a positive fashion.

3. Conducted a mutant screen in field grown soybeans. The mineral content of soybeans is an important component of the human diet. ARS scientists developed a method of screening large, field-grown mutant populations of soybeans for lines with altered elemental profiles. They tested several methods of sorting through the data and found the most efficient computational method to identify potential mutant lines, which were then regrown to confirm the predictions. They demonstrated that the chosen method is successful at identifying mutant lines. This computational method can therefore be used to identify genes used to produce lines with improved nutritional properties, an important goal of U.S. plant breeders and one that impacts the competitive health of the soybean industry.

4. Flow of carbon and nitrogen into protein in developing soybeans. Soybeans store approximately 40% of their biomass in the form of protein. Protein concentration reflects the carbon and nitrogen levels received by the developing embryo, but the correlation between the two is poorly understood even though it is of major importance in soybean seed improvement. The relationship between carbon and nitrogen supply and seed composition during filling was examined through a series of plant embryo culturing experiments by investigators in St Louis, MO. The analyses revealed that protein concentration can be manipulated by nitrogen supply and is variable but protein components do not change with nitrogen supply. When nitrogen is supplied in the form of an amino acid (glutamine), the co-introduced carbon is distributed between new amino acid synthesis and the synthesis of fatty acids that ultimately get stored as oil. The results also indicated that seed metabolism can accommodate different levels of protein biosynthesis while maintaining a consistent rate of dry weight. This work lends insight to scientists that are engineering a soybean with altered composition.

5. Probing subcellular metabolism in seeds. Plants utilize a basic metabolism that features substantial duplication of metabolite pools and enzyme reactions in different subcellular compartments for growth and development. This metabolic duplication poses a significant challenge for understanding how plant processes are regulated and how they can be manipulated for crop improvement. ARS scientists in St. Louis, MO explored the extent to which amino acids (one group of metabolites) are made in individual cellular compartments. Scientists used a variety of non-radioactive labeled (13Carbon) compounds to track where metabolic events that generate proteins occur in different locations within the cells of the soybean embryo (isolated from the seed). When particular 13C amino acids were used to feed the embryos the label appeared preferentially in chloroplasts, a major organelle in plant cells. Some amino acids are thus preferentially channeled into one compartment and are not evenly distributed within cells. The use of this approach and the results it generates have importance in understanding seed metabolism and for developing more rational metabolic engineering of seed composition for novel and established agricultural needs.


Review Publications
Zhao, J., Wang, C., Welti, R., Bedair, M., Sumner, L., Baxter, I.R., Wang, X. 2011. Suppression of phospholipase D,gammas confers increased aluminum resistance in Arabidopsis thaliana. PLoS One. 6(12):e28086. Available: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0028086.

Tjellstrom, H., Yang, Z., Allen, D.K., Ohlrogge, J.B. 2011. Rapid kinetic labeling of Arabidopsis cell suspension cultures: Implications for models of lipid export from plastids. Plant Physiology. 158:601-611.

Baxter, I.R., Dilkes, B. 2012. Elemental profiles reflect plant adaptations to the environment. Science. 336:1661-1663.

Allen, D.K., Laclair, R.W., Ohlrogge, J.B., Shachar-Hill, Y. 2012. Isotope labeling of rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments. Plant Cell and Environment. 35(7):1232-1244.

Baxter, I.R., Hermans, C., Lahner, B., Yakubova, E., Tikhonova, M., Verbruggen, N., Chao, D., Salt, D.E. 2012. Biodiversity of mineral nutrient and trace element accumulation in Arabidopsis thaliana. PLoS One. 7(4):e35121. Available: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0035121.

Last Modified: 7/25/2014
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