Summary<br>
(1)&nbsp;&nbsp; Senior personnel<br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; PI:&nbsp;&nbsp; 
Alan Myers, Department of Biochemistry, Biophysics, and Molecular Biology, Iowa 
State University<br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Co-PI:&nbsp;&nbsp; Martha James, Department 
of Biochemistry, Biophysics, and Molecular Biology, Iowa State University<br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; Co-PI:&nbsp;&nbsp; Eve Wurtele, Department 
of Botany, Iowa State University<br>
(2)&nbsp;&nbsp; Summary of the proposed project<br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; The molecular and physiological functions 
of the Arabidopsis starch metabolism gene network will be determined.&nbsp; Carbohydrate 
storage as starch is a distinguishing characteristic of plants.&nbsp; The glucose 
homopolymers in starch, amylose and amylopectin, comprise monomers joined by a(1&reg;4) 
or a(1&reg;6) glycosidic bonds.&nbsp; Despite this simple chemical structure, 
amylopectin in particular displays a complex molecular architecture essential 
for starch function.&nbsp; Mechanisms for amylopectin biosynthesis or degradation 
to soluble glucose monomers are largely unknown.&nbsp; The Arabidopsis genome 
sequence allows identification of all the genes involved in starch biosynthesis 
or mobilization, and examination of each function individually and within the 
metabolic network.&nbsp; <br>
&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp; The study focuses on 28 genes, those 
involved after the production of the glucosyl unit donor.&nbsp; The gene set includes 
starch synthases, branching enzymes, debranching enzymes, a-amylases, b-amylases, 
disproportionating enzymes, and starch phosphorylases.&nbsp; In each instance 
the genome sequence predicts multiple isoforms.&nbsp; We propose that most isoforms 
have specific roles in creating or dismantling the molecular architecture of starch, 
and that many components of the network act via direct functional interactions, 
rather than in a series of independent enzymatic steps.&nbsp; The specific aims 
are as follows.<br>
&nbsp;&nbsp;&nbsp; 1)&nbsp;&nbsp; Tools will be created, including (a) mutants 
defective for each gene in the network, (b) isoform-specific antibodies for each 
protein, and (c) E. coli strains overexpressing each protein.<br>
&nbsp;&nbsp;&nbsp; 2)&nbsp;&nbsp; Starch biosynthesis and degradation will be 
characterized in each mutant via structural and metabolic analyses.<br>
&nbsp;&nbsp;&nbsp; 3)&nbsp;&nbsp; Effects of each mutation on the complement of 
specific enzyme isoforms will be determined using high-resolution, two-dimensional 
zymograms.<br>
&nbsp;&nbsp;&nbsp; 4)&nbsp;&nbsp; Proteins in the study set will be tested for 
physical interaction with other components of the network.<br>
&nbsp;&nbsp;&nbsp; 5)&nbsp;&nbsp; The tissues in which each gene is expressed 
will be identified.<br>
&nbsp;&nbsp;&nbsp; 6)&nbsp;&nbsp; Effects of each mutation on expression of other 
Arabidopsis genes will be examined by global mRNA profiling.


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