Seed storage reserves represent one of the most important sources of renewable fixed carbon and nitrogen found in nature. Seeds are well-adapted for diverting metabolic resources to synthesis of storage proteins as well as enzymes and structural proteins needed for their transport and packaging into membrane bound storage protein bodies.<P> Our underlying hypothesis is that the ER stress response provides the critical cellular control of metabolic flux required for optimal accumulation of storage reserves in seeds. This highly conserved response is a cellular mechanism to monitor the protein folding environment of the ER and restore homeostasis in the presence of unfolded or misfolded proteins. In seeds, deposition of storage proteins in protein bodies is a highly specialized process that takes place even in the presence of mutant proteins that no longer fold and package properly.<P> We will focus on the means by which the ER must sense and respond to functional perturbations from the synthesis and trafficking of proteins to the endomembrane. We hypothesize that the ER stress response in plants is mediated through multiple pathways involving IRE1, lipid metabolism, selective translational attenuation and ER associated protein degradation.<P> Links between IRE1, lipid metabolism and ER stress have been established; here we propose three specific aims to test the remaining elements of our hypothesis: <BR>We will determine whether misfolded proteins are degraded in cell cultures and maize endosperm mutants. <BR> A corollary of our hypothesis suggests that a common sensing mechanism should activate the individual branches of the ER stress response. We will use transient association with the molecular chaperone BiP as an affinity tag to identify signal transduction proteins for ER stress. <BR> To determine if protein bodies retain the key components that regulate cellular homeostasis during ER stress, we will compare the protein subunits of multichaperone complexes in ER and protein bodies of normal seeds and ER stressed mutants. <BR>Protection of the seed from pathogen damage is achieved in part by production of anti-fungal compounds. <P>We will identify maize kernel compounds that perturb aflatoxin biosynthesis and development in Aspergillus flavus and therefore can be selected for as traits in resistance breeding. Improving agronomic quality of seed crops is an important and multi-faceted problem involving, in part, gene expression, protein synthesis and efficient compartmentalization of proteins within membranes of seeds. To alter these concerted events will require not only an understanding of the metabolic pathways involved but also the ability to predict the impact of genetic alterations on lipid and protein biosynthesis, and protein body formation. <P>The studies outlined here will increase our knowledge of regulation of crucial metabolic pathways of seeds and provide a framework for increasing and improving renewable energy resources.
Non-Technical Summary: Before we can exploit and manipulate energy reserves of soybean, maize or other crop species, we must understand the mechanisms by which seed protein and lipid biosynthetic pathways are coordinated and regulated. Understanding the regulation of crucial metabolic pathways will provide a framework for increasing biomass production and improving renewable energy resources. We and our collaborators have shown a positive correlation between the presence of misfolded proteins and cellular responses to increase the folding capacity of the cell and accelerate protein degradation. These responses are spatially segregated as though the stress response in the endoplasmic reticulum does not always alter functions in the entire organelle but is instead specific to the site of the processing and deposition of the altered protein. This information has increased our knowledge of the regulation of crucial metabolic pathways of seeds and should provide a framework for increasing and improving renewable energy resources. In addition, our identification of components of the protein degradation machinery may enable the improvement of expression of genetically-engineered proteins and the amelioration of grain quality problems such as endoplasmic reticulum associated degradation of foreign proteins. Production of aflatoxin by Aspergillus flavus is a limiting factor for corn cultivation in southern growing regions. Identification of preformed resistance proteins to inhibit fungal growth or toxin production would be an important step toward development of strategies for control of fungal pathogens. <P> Approach: 1. We will determine whether translation is attenuated and/or misfolded proteins are degraded in cell cultures and maize endosperm mutants. We have developed probes and characterized maize endosperm mutants that allowed us to demonstrate links between ER stress, molecular chaperone induction and induction of phospholipid biosynthetic enzymes. We will carry out a rigorous investigation of these ER stress responses, focusing on links to ERAD under "chronic" ER stress conditions (endosperm mutants) and "acute" stress conditions (caused by chemical induction). 2. A corollary of our hypothesis suggests that a common sensing mechanism should activate the individual branches of the ER stress response. We will use transient association with the molecular chaperone BiP as an affinity tag to identify signal transduction proteins for ER stress. The diversity of downstream effects we have observed during ER stress demonstrates a complex response. We will take advantage of the apparent function of BiP as a central sensor of ER stress that binds to transmembrane signaling molecules in the absence of unfolded proteins and releases them to initiate the ER stress response as it associates more tightly with unfolded targets. We will identify such sensors of ER stress by their association with BiP in unstressed cells and lack of association in ER-stressed cells. Use of maize endosperm mutants gives us the advantage of having large enough quantities of experimental material to enrich the recovery of BiP found in association with membrane bound transducer molecules. 3. To determine if protein bodies retain the key components that regulate cellular homeostasis during ER stress, we will compare the protein subunits of multichaperone complexes in ER and protein bodies of normal seeds and ER stressed mutants. We will investigate the higher order complexes of molecular chaperones that have been found in both stressed and unstressed cells to determine whether they, like membranes, have undergone changes as ER membranes are transformed into protein body membranes. 4: To assess the antifungal activity of chitinase (ChiA) and zeamatin (ZLP) proteins we first plan to separate them using a chitin affinity column to 1) confirm if the ChiA protein we have recovered does in fact have chitin-binding activity and 2) separate ChiA and ZLP to determine if only one of the two proteins is responsible for the majority of the growth inhibition. We will assay the separated proteins independently and in combination. We will produce ChiA and ZLP recombinantly to verify that growth inhibition is not due to some other protein. Recombinant protein will also be used for antibody production for screening of germplasm. Growth inhibition will be tested in microtiter plate A. flavus bioassays. In addition, we will determine if these genes are induced in response to A. flavus infection beginning with microarray data from a field-infection study recently completed.