Source: VIRGINIA POLYTECHNIC INSTITUTE submitted to
SOYBEAN CROP IMPROVEMENT: ENHANCING NUTRIENT UTILIZATION
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0199805
Grant No.
(N/A)
Project No.
VA-135722
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jul 1, 2004
Project End Date
Jun 30, 2009
Grant Year
(N/A)
Project Director
Grabau, E. A.
Recipient Organization
VIRGINIA POLYTECHNIC INSTITUTE
(N/A)
BLACKSBURG,VA 24061
Performing Department
PLANT PATHOLOGY PHYSIOLOGY & WEED SCIENCE
Non Technical Summary
The presence of high phosphorus levels in animal waste contributes to environmental phosphorus pollution. This purpose of this project is to improve nutrient availability in soybean by reducing seed phytate levels.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2041820100015%
2041820104015%
2041820108020%
2061820100015%
2061820104015%
2061820108020%
Goals / Objectives
The long-term goal of the project is to improve the value of soybean by lowering the phytate content in seeds. Reduced phytate will have nutritional benefits and provide livestock producers with an additional tool for phosphorus waste management. Two strategies for reducing seed phytate include introducing phytase gene to degrade phytate as it accumulates and blocking phytate synthesis in developing seeds.
Project Methods
Reduction of seed phytate will be pursued by two approaches: 1) by engineering soybean plants for expression of a soybean phytase during seed development, and 2) by altering the phytate biosynthetic pathway. Transgenic soybean plants will be analyzed for phytase gene expression, phytase protein localization, enzyme activity and seed phytate levels. Optimal timing of expression will be examined by use of different promoters. Protein localization will be investigated by generation of fusion proteins with GFP or epitope tags, use of specific targeting sequences, and dection of subcellular localization by immunodetection. To alter the phytate biosynthesis, the pathway will be elucidated in soybean. Genes encoding critical pathway steps will be isolated and an RNAi approach used to altering expression of pathway genes.

Progress 07/01/04 to 06/30/09

Outputs
OUTPUTS: Outputs and activities for this project aimed at improving nutrient availability and utilization in soybean include research to develop soybeans with low phytic acid (myo-inositol hexakisphosphate) content. To achieve this we have used gene identification, cloning and expression strategies to characterize key enzymes in the soybean phytic acid biosynthetic pathway. We have added a goal of characterizing transporters putatively involved in the low phytate trait. We are utilizing RNA interference protocols to reduce several candidate kinases involved in the accumulation of low phytate in soybeans. We have presented results at conferences and published results in scientific journals. We have trained doctoral students and mentored undergraduate students in independent research projects. PARTICIPANTS: Elizabeth Grabau, PI; Amanda Stiles, graduate student; Saghai Maroof, collaborator; Javad Torabinejad, previous collaborator TARGET AUDIENCES: Target audiences include scientists and students in the areas of plant science, nutrition and environmental sustainability. Low phytate soybean as a product of this work will be valuable to the livestock industry for reducing cost via increased nutrient availability and reducing environmental pollution by eliminating excess phosphorus excretion and potential for run-off and eutrophication. PROJECT MODIFICATIONS: No major changes during the project.

Impacts
The results of the research have contributed to a new understanding of phytic acid biosynthesis and the basis for the low phytate trait in soybeans. Low phytate soybeans will provide livestock producers with better nutrient management strategies, reduce cost of animal diets and reduce environmental phosphorus pollution associated with livestock industries. Diets composed of low phytate feed components will provide better mineral nutrition, reduce phosphorus excretion, prevent run-off and the potential for eutrophication of surface waters. Characterization of the inositol phosphate kinase enzymes helped elucidate the pathway for phytic acid. Genetic studies to characterize low phytate lines have demonstrated the potential involvement of ABC transporters with the low phytate trait.

Publications

  • Saghai Maroof, M.A., Glover, N., Biyashev, R., Buss, G., Grabau, E.A. 2009. Genetic basis of the low-phytate trait in the soybean line CX1834. Crop Sci. 49: 69-76.


Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: Goals are to reduce soybean seed phytate for improved phosphorus availability. Phytate (InsP6) in animal diets is poorly utilized and results in phosphorus pollution associated with livestock production. To reduce synthesis and accumulation, the biosynthetic pathway for phytate is being investigated. We have identified gene families for important kinase enzymes involved in phytate synthesis in soybean. Gene expression and enzyme purification studies have provided information on the biochemical characterization of the pathway. We continue to collaborate with Dr. Saghai Maroof in the Department of Crop and Soil Environmental Sciences to study low phytate soybean lines identified through mutagenesis. Research results have been disseminated as peer-reviewed publications (see below and one paper in press). I have received an invitation to write a review article on one of the inositol kinase gene families previously reported. PARTICIPANTS: My laboratory collaborates with Dr. Saghai Maroof in the the Department of Crop and Soil Environmental Sciences at Virginia Tech. We have investigated the nature of mutations in low phytate soybean lines and have a Crop Science paper currently in press. Dr. Torabinejad in PPWS at Virginia Tech was a collaborator on the tissue culture aspects of this project. Ms. Haijie Liu has a 50% appointment to my program and has been working on construction of transformation plasmids. TARGET AUDIENCES: Target audiences include researchers and students in the plant sciences interested in inositol phosphate metabolism, nutritionists and animal scientists interested in nutrient availability, and environmental scientists interested in reducing phosphorus pollution. Change in knowledge about seed phytate and the ability to modify and reduce its abundance will provide opportunities for better nutrition, improved phosphorus availability in animal diets and reduced phosphorus pollution associated with livestock production. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
OUTCOMES. We have initiated embryogenic tissue culture lines from normal and low phytate soybean cultivars to invesitgate the synthesis of phytate in culture. Labeling with 3H-inositol confirms reduced synthesis in the low phytate lines. We are utilizing an RNA interference approach to reduce phytate biosynthesis. Transformation plasmids containing the RNAi cassettes for three kinase genes have been constructed. Collaboration with Dr. Saghai Maroof has yielded a molecular characterization of mutation believed responsible for the low phytate phenotype. IMPACT. Elucidation of phytate biosynthesis will allow us to manipulate enzyme expression to reduce phytate production in seeds. Low phytate cereals and legumes will provide better phosphorus availability and reduce environmental phosphorus pollution and the resulting potential for run-off and eutrophication. It will also improve mineral nutrition by reducing the amount of binding of essential dietary minerals such as iron and zinc to the highly charged phytate. The effects of reduced phytate will have a positive environmental impact and improved nutritional availability in populations consuming legumes and cereals as dietary staples.

Publications

  • Stiles, A.R., Qian, X., Shears, S.B. and Grabau, E.A. 2008. Metabolic and Signaling Properties of an Itpk Gene Family in Glycine max. Febs Lett. 582:1853-1858.


Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: The goals and objectives of this project are to improve the value of soybean by lowering seed phytate content. Phytate (also known as myo-inositol hexakisphosphate or IP6) in animal diets is unavailable to non-ruminants. Improving phytate phosphorus availability will reduce environmental phosphorus pollution associated with livestock production. Reducing phytate accumulation requires an understanding of the biosynthetic pathway for phytate. We have isolated and characterized several kinase genes or gene families involved in inositol phosphate metabolism to determine their roles in phytate biosynthesis. We have focused on myo-inositol (1,4,5)P3 6/3-kinase, myo-inositol(1,3,4)P3 5/6-kinase (family of four genes), and myo-inositol(1,3,4,5,6)P5 2-kinase. We have examined gene expression patterns during seed development to demonstrate likely participants in seed phytate synthesis. We have generated tagged fusion proteins for expression in E. coli and purification of the proteins by affinity chromatography. We have utilized the fusion proteins to characterize the substrates, enzyme activities and kinetics. We have utilized complementation in yeast to confirm enzyme function. PARTICIPANTS: Amanda Stiles, Graduate Student, Dept. of Plant Pathology, Physiology and Weed Science, Virginia Tech. Dr. Stiles pursued this project as part of her doctoral research and received her Ph.D. in August 2007. Javad Torabinejad, Research Assistant Professor, Dept. of Plant Pathology, Physiology and Weed Science, Virginia Tech. Dr. Torabinejad was a collaborator on this project and continues involvement in construction of gene silencing vectors. TARGET AUDIENCES: Target audiences are: 1) researchers and students in plant sciences interested in various aspects of plant inositol phosphate metabolism, both the gene expression and biochemistry, 2) animal scientists interested in nutrient availability for non-ruminants, including humans, and 3) environmental scientists interested in reducing environmental phosphorus pollution

Impacts
Studying the phytate biosynthetic pathway will provide the ability to target individual steps in the pathway for down-regulation to reduce phytate synthesis in soybean seeds. We have targeted the late steps in the pathway for down-regulation by gene silencing. The impact of reduced phytate content will be to improve phosphorus availability for non-ruminant animals consuming soy-based diets. This will provide additional waste management tools to livestock producers to reduce phosphorus content in manure and croplands to which manure is applied as fertilizer. This in turn will reduce the possibility of phosphorus pollution of surface waters and accompanying eutrophication from excess nutrients. Additional impacts will result from the improved availability of essential mineral nutrients that are normally bound to phytate, such as iron and zinc. Populations that consume legumes and cereals as dietary staples can experience mineral deficiencies associated with the phytate content in these foods.

Publications

  • Gao, Y., Shang, C., Saghai Maroof, M.A., Biyashev, R.M., Grabau, E.A., Kwanyuen, P., Burton, J.W. and Buss, G.R. 2007. A modified colorimetric method for phytic acid analysis in soybean. Crop Science 47: 1797-1803.
  • Chamberlain, P.P., Qian, X., Stiles, A.R., Cho, J. Jones, D.H., Lesley, S.A., Grabau, E.A., Shears, S.B. and Spraggon, G. 2007. Integration of inositol phosphate signaling pathways via human ITPK1. J. Biological Chemistry 282: 28117-28125.
  • Chiera, J.M. and Grabau, E.A. 2007. Localization of myo-inositol phosphate syntase (GmMIPS-1) during the early stages of soybean seed development. J. Experimental Botany 58: 2261-2268.
  • Stiles, Amanda. 2007. Identification and characterization of late pathway enzymes in phytic acid biosynthesis in Glycine max. Ph.D. Dissertation, Virginia Polytechnic Institute and State University. 117 pages.


Progress 10/01/05 to 09/30/06

Outputs
Further modification of seed phytate content will benefit from a better understanding of the biosynthetic pathway. To elucidate the pathway in soybean we have identified and sequenced genes for enzymes that act at six different steps in the pathway, including myo-inositol 3-phosphate synthase (MIPS), myo-inositol kinase (MIK), inositol monophosphatase (IMP), myo-inositol (1,3,4)P3 5/6-kinase (I5/6K), myo-inositol (1,4,5,)P3 6/3-kinase (I6/3K), and myo-inositol (1,3,4,5,6)P5 2-kinase (I2K). We have expressed these genes as tagged fusion proteins in bacteria and purified the proteins by affinity chromatography for characterization of the biochemical properties. We are generating plasmid constructs for targeting the down-regulation of these genes by RNA interference, which is expected to reduce phytate synthesis.

Impacts
Improving soybeans for reduced phytate content will provide better nutrient utilization in human and animal diets, reduce phosphorus content in manure, reduce entry of excess phosphorus into soil and water, and provide livestock producers with better tools for nutrient management.

Publications

  • Lei, X., Blake, J.P., Forsberg, C.W., Fox, D.G., Grabau, E., Mroz, Z., Sutton, A.L., Walker, W.R., and Webb, K. 2006. Biotechnological approaches to manure nutrient management, Animal agricultures future through biotechnology, part 4, Council for Agricultural Science and Technology Issue Paper 33: 20 pgs.


Progress 10/01/04 to 09/30/05

Outputs
Transgenic soybeans were generated that contained a soybean phytase gene under control of the soybean beta-conglycinin promoter for seed specific expression. Transformants were analyzed for transgene copy number and inheritance. RNA expression and enzyme activity were examined in developing seeds and phytate content was determined in mature soybean seeds. Soybean plants with low phytase transgene copy numbers were propagated for four generations. Phytase transgene expression was similar to expression of beta conglycinin during seed development as expected from the promoter used in the gene construct. Phytase activity levels in developing embryos were elevated in transgenic plants compared to non-transformed control soybeans. Activity increased linearly in seeds from 5 to 9 mm in size. Seed phytate levels were reduced from 12.6 to 24.8 percent for four transgenic lines. Available phosphorus was increased approximately 3-fold in phytase-expressing soybeans.

Impacts
Use of gene engineering to reduce soybean seed phytic acid levels will allow more efficient nutrient utilization in animal diets, reduced environmental phosphorus pollution from intensive livestock production, and improved human mineral nutrition.

Publications

  • Chiera, J.M., Finer, J.J. and Grabau, E.A. 2004. Ectopic expression of a soybean phytase in developing seeds of Glycine max to improve phosphorus availability. Plant Molecular Biology 56: 895-904.


Progress 10/01/03 to 09/30/04

Outputs
Project objectives are to engineer soybean plants for reduced seed phytate content utilizing multiple approaches. The two strategies to be employed include degrading accumulating phytate via the expression of a developmentally-regulated phytase and blocking phytate synthesis using gene silencing technologies. A soybean phytase has been isolated and introduced under control of a seed specific promoter into soybean tissue culture by gene gun bombardment. Transgenic soybeans expressing the phytase have been characterized to analyze gene copy, RNA expression and enzyme activity. Phytate and corresponding available phosphorus levels have been analyzed by ferric chloride precipitation and high performance liquid chromatography (HPLC). Phytate (IP6) has been reduced up to 19 percent in transformed plants. Several candidate genes in the phytic acid biosynthetic pathway have been identified, including myo-inositol 3-phosphate synthase, myo-inositol (1,3,4)P3 5/6-kinase, myo-inositol (1,4,5)P3 3/6-kinase, and myo-inositol (1,3,4,5,6)P5 2-kinase. The genes have been cloned into bacterial expression vectors for protein production and purification by affinity chromatography. Enzyme activity has been confirmed for the Ins(1,3,4)P3 5/6-kinase and Ins(1,4,5)P3 3/6-kinase. Further biochemical characterization of the enzymes is underway.

Impacts
Use of gene engineering to reduce soybean seed phytic acid levels will allow more efficient nutrient utilization in animal diets, reduced environmental phosphorus pollution from intensive livestock production, and improved human mineral nutrition.

Publications

  • Chiera, J.M., Finer, J.J., and Grabau, E.A. 2004. Ectopic expression of a soybean phytase in developing seeds of Glycine max to improve phosphorus availability. Plant Molecular Biology (in press).