Characterization and Modification of a Biosynthetic Pathway in Glycine max

1. Characterization and Modification of a Biosynthetic Pathway in Glycine max Improving the nutritional characteristics of soybeans Stiles AR, Grabau EA, astiles@vt.edu Department of Plant Pathology, Physiology and Weed Science Virginia Polytechnic Institute and State University, Blacksburg, VA 24061 Gene Expression of Late Pathway Kinases in Developing Seeds Project Goals • Identify and characterize genes in the phytate biosynthetic pathway in soybean. • Clone, purify, and characterize each soybean kinase enzyme using a bacterial expression system. • Generate silencing vectors to allow individual down-regulation of each kinase gene in embryogenic soybean culture using RNA interference (RNAi) technology. seed size (mm) 9-10 0-4 5-6 7-8 B A A1 Phytate or myo-inositol hexakisphosphate is the major storage form of phosphorus present in plant seeds. Bound phytate phosphorus and chelated mineral cations are largely unavailable in the diets of non-ruminants due to a lack of digestive enzymes to remove phosphate groups. Phytate phosphorus is excreted in manure, accumulates in soil, and eventually enters watersheds. As a result, phytate is considered both an antinutrient and an environmental pollutant. Altering seed composition to reduce phytate, improve nutrient availability, and reduce phosphorus pollution will require an understanding of the phytate biosynthetic pathway. We have isolated several myo-inositol phosphate kinase genes in soybean as possible candidates for steps in the biosynthetic pathway. We have characterized the genes for four myo-inositol (1,3,4)P3 5/6-kinases, one myo-inositol(1,4,5)P3 6/3/5-kinase, one myo-inositol(1,3,4,5,6)P5 2-kinase and one myo-inositol kinase. We have examined expression in developing seeds and other tissues by Northern blot analysis and quantitative RT-PCR. We have expressed all seven genes as GST fusion proteins in E. coli, and verified enzyme activity on the proposed substrates. We are conducting biochemical characterization to determine enzyme kinetics and substrate specificities. We are utilizing soybean embryogenic cultures to test for alterations in inositol phosphate profiles as a result of down-regulating the kinase genes by RNA interference. A2 A3 Gene Identification A4 • 7 full length coding regions were identified and amplified from soybean mRNA. • 1 myo-inositol kinase gene (MIK) • 4 Ins(1,3,4)P3 5/6-kinase genes (A1 – A4) • 1 Ins(1,4,5)P3 6/3/5-kinase gene (B) • 1 Ins(1,3,4,5,6)P5 2-kinase gene (C) B C The Phytate Problem Figure 2. A) Northern Blots of RNA from developing soybean seeds. Total RNA was extracted from soybean seeds of 0-4 mm, 5-6 mm, 7-8 mm, and 9-10 mm. The blots were probed with full length cDNA sequences and detected using chemiluminescence. B) Real-time PCR results quantifying mRNA expression levels of genes A1-A4, B and C in developing seeds. Reverse primers were generated from the 3’UTR region of each gene for specificity. Expected Size (kD) Protein Protein ID Amino Acids Protein Expression and Characterization Ins(1,3,4)P3 5/6-kinase A1 339 38.5 A2 315 35.0 A3 354 40.1 A4 341 38.4 Ins(1,4,5)P4 6/3/5- kinase B 279 30.9 The phytate molecule chelates minerals. Ins(1,3,4,5,6)P5 2-kinase C 456 51.2 Table 1. Table of the predicted protein masses based on the amino acid coding region for each protein (GST tag region not included). Soybeans store phosphorus and minerals in their seeds in the form of phytate. Figure 3. SDS-PAGE gels showing the purification of GST-fusion proteins (A) Proteins A1-A4 (B) Protein B. Lane 1 in each gel contains the Mark-12 protein size marker (Invitrogen). Biosynthetic Pathway Activity Assays Each protein was incubated with its proposed substrate and the products analyzed using High Performance Liquid Chromatography (HPLC). The excess phosphorus is released into the environment where it contributes to phosphorus pollution. Soybeans are one of the main components of livestock feed. However, non-ruminant animals cannot break down the phytate molecule. They are unable to use the phosphorus and minerals stored in phytate, and farmers must supplement their feed. Harmful effects of excess phosphorus runoff into a pond. Phytate passes through the animal…. Figure 4. HPLC elution profile of the products of the Ins(1,4,5)P3 6/3/5-kinase assay. (A) Trace showing the IP3 and IP4 standards. (B) Trace showing the products of the B activity assay. Figure 5. HPLC elution profiles of the products of the Ins(1,3,4)P3 5/6-kinase activity assays. (A) Trace showing the control reaction lacking enzyme. (B – E) Traces showing the products of the A1-A4 activity assays. Into the soil and groundwater where it is broken down by bacteria. Figure 1. Two predicted pathways for phytic acid biosynthesis. Figure modified from Raboy (2001). Our Goal References Studies conducted in various organisms have demonstrated two potential routes for the phosphorylation of myo-inositol; a phosphatidylinositol (PtdIns)-dependent and a PtdIns-independent pathway. In yeast, the PtdIns-dependent pathway includes an Ins(1,4,5)P3 6/3/5-kinase. In Zea mays an Ins(1,3,4)P3 5/6-kinase has been identified that is characteristic of an independent pathway [1]. A mutation in this Zea mays 5/6-kinase gene creates a low phytic acid (lpa) phenotype. In Arabidopsis, genes encoding both kinases have been identified [2,3]. 1. Shi, J., Wang H., Wu Y., Hazebroek J., Meeley R., Ertl D. (2003) The Maize Low-Phytic Acid Mutant lpa2 Is Caused by Mutation in an Inositol Phosphate Kinase Gene. Plant Physiol. 131:1-9. 2. Wilson, M.P., Majerus, P.W. (1997) Characterization of a cDNA Encoding Arabidopsis thaliana Inositol 1,3,4-trisphosphate 5/6-Kinase. BBRC 232: 678-681. 3. Stevenson-Paulik J., Odom A.R, York J.B. (2002) Molecular and Biochemical Characterization of Two Plant Inositol Polyphosphate 6-/3-/5- Kinases. J. Biol Chem. 277: 42711-42781. To understand how the soybean plant synthesizes phytate. We can then work to develop a plant with decreased levels of phytate and higher levels of available phosphorus. This will generate a more nutritious soybean and decrease phosphorus pollution from livestock farms.