Recipient Organization
IOWA STATE UNIVERSITY
2229 Lincoln Way
AMES,IA 50011
Performing Department
(N/A)
Non Technical Summary
Starch is an agriculturally important commodity and renewable raw material. Starch utility stems from structural features of the most abundant polymer in starch, amylopectin (Ap). Throughout Ap, linear chains of glucose units are periodically branched. Enzymes that catalyze formation of linear chains are starch synthases (SS), and those that introduce branches are starch branching enzymes (BE). Debranching enzymes (DBEs) also have a role. This research seeks to understand how SSs, BEs, and DBEs are coordinated and regulated to produce the architectural arrangement of branch chains in AP of maize, an important starch-producing crop plant. The central hypothesis is that SSs, BEs, and DBEs are regulated in part by direct physical interactions with each other and with other proteins. Biochemical evidence for protein complex formation and specific protein-protein binding will be sought. Objectives are: 1) characterization of maize DBEs in terms of molecular size, enzyme
activity, and co-fractionation with other starch enzymes; 2) analysis of the effects of DBE mutations on other enzymes activities; and 3) investigation of the effects of altered DBE expression on other maize proteins, using state-of-the-art proteomics technologies. Starch is the major carbohydrate component of most diets, and is widely used in food and industrial processing. As understanding of starch biosynthesis increases, so does the potential to modify starch in the plant in ways that can expand starch usage.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
The goal of the research is to understand the regulation and coordination of debranching enzyme (DBE), branching enzyme (BBE) and starch synthase (SS) activities in the determination of starch structure. Native forms of two maize endosperm DBEs, SU1 and ZPU1, will be characterized in terms of their molecular mass and co-fractionation with other starch biosynthetic enzyme activities. Pleiotropic effects of DBE mutations and transgenic alterations in DBE function on the activities of other starch metabolizing enzyme isoforms will be determined in starch zymograms. Allele-specific effects will be identified. The effects of altered DBE function on the protein composition in plastids of developing kernels will be examined using proteomics technology.
Project Methods
The multimeric state of the DBEs as they exist in maize endosperm will be characterized. SU1 and ZPU1 will be partially purified from developing kernels by gel permeation chromatography (GPC) and/or anion exchange chromatography (AEC). Fractions containing DBE and other starch metabolizing activities will be identified by enzymatic assay and immunoblot analysis using antibodies that specifically recognize SSs, BEs, and DBEs. Mutant analysis will be applied if indicated, through analysis of chromatography profiles of DBEs, SSs, and BEs mutants. Unknown polypeptides that co-fractionate with DBEs will be purified in a series of chromatography steps and identified by mass spectroscopic analysis using MALDI-TOF technology and database comparison of molecular weights of the tryptic peptides. Pleiotropic effects of DBE genetic mutants and transgenic plants on the activities of starch metabolizing isoforms will be investigated using starch zymogram analysis of mid-development
endosperm proteins. One-dimensional zymograms will analyze crude protein extracts and 2-D zymograms will analyze proteins fractionated by AEC. The effects of altered DBE expression of on the protein composition in plastids of developing kernels will be examined using proteomics technology to identify any polypeptide that changes in abundance, isoelectric point, or apparent molecular weight. Protein mixtures will be subjected to high resolution isoelectric focusing separations in the first dimension using immobilized pH gradients. The 2-D separations will be accomplished by standard SDS-PAGE performed using an apparatus optimized for high reproducibility and the 2-D pattern of spots will be analyzed using computer that compares stained gel images. Identification of proteins that are altered in the su1 or zpu1 mutants will be accomplished by both immunological and mass spectrometric methods.