1a. Objectives (from AD-416):
To enhance knowledge of the synthesis and role of diphospho- and tri-phospho inositol phosphates during plant and seed development.
1b. Approach (from AD-416):
Diphospho- and triphospho-inositol phosphates will be characterized in plant and seed tissues during development and in response to abiotic stress. Genes and enzymes that are important to diphospho- and triphospho-inositol phosphate metabolism will be characterized. Work would primarily involve maize, barley and the model system Arabidopsis. Genomic and cDNA sequences encoding a target enzyme would be cloned and recombinant proteins would be expressed using a heterologous system like E. coli. The recombinant proteins would be used to characterize each enzymes’ biochemical properties. This includes substrate specificity and catalytic property/inhibitor-effector analyses. Mutations in selected genes will be isolated using the forward genetics approach called “TILLING”. Plant homozygous for mutations will be studied to determine the role and importance of genes.
3. Progress Report:
This work contributes to National Program 301, Component 2, Crop Informatics, Genomics and Genetic Analyses, Problem 2C, Genetic Analyses and Mapping of Important Traits. The traits in question concern the nutritional quality of seed crops. The parent project focuses on the genetics of plant and seed phosphorus and phytic acid. Both the total amount of phosphorus and its chemistry are critically important to the end-use quality of grain and legume crops. This project is part of an NSF-funded cooperation between the USDA-ARS, North Carolina State University and Virginia Tech that focuses on a novel class of compounds in plants, the diphospho- and triphospho-inositol phosphates. These compounds are believed to be important to “metabolic status sensing” which plays a part in how plants sense and respond to stress such as cold stress or drought stress, and are also important to nutrient utilization and seed chemistry. The three main objectives of the USDA-ARS component of this project are: 1) the development of analytical methods useful for this work; 2) the purification of these compounds from plant tissues and their structural characterization, and 3) genetics of these compounds in maize, including isolation of recessive alleles of important genes and genetic screens for mutations that alter the level of these compounds in seeds. Progress has been made in each of these objectives. For Objective 1, maintenance and upgrade of equipment and technologies needed for this work were completed in this year. For Objective 2, new populations of maize that produce seed ideal for the purification of these compounds were produced in a winter nursery and increased amounts of a valuable Arabidopsis genotype were produced, and the first efforts at purification and structural characterization are currently underway. For Objective 3, two key genes were identified in maize and isolation of alleles of these genes is underway. Also, methods for screening for novel mutations that alter these compounds were established and trial runs were conducted. Communication between cooperators on this project is via conference calls, the internet, and e-mail communications. A video conference for all participants held on 5/30/2012.