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.
This work contributes to National Program 301, Component 2, Crop Informatics, Genomics and Genetic Analyses, Problem 2C, Genetic Analyses and Mapping of Important Traits and supports objective 3 of the parent project. The traits in question concern the nutritional quality of seed crops. This 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 in turn is important to abiotic stress response, to nutrient utilization, and seed chemistry. The main objective of the USDA-ARS component of this project is the development of analytical methods useful for this work, the purification of these compounds from plant tissues, their structural characterization, and genetic screening for mutations that alter the level of these compounds in seeds. Initial progress has been made in each of these objectives. Populations of maize that produce seed ideal for the purification of these compounds are currently being grown in summer nurseries. Routine methodology for their detection and quantitation are under development. An initial breakthrough concerns maize lpa1, which encodes a transport function important to the storage of a precursor of these compounds, inositol hexaphosphate or Ins P6 (“phytic acid”). The USDA-ARS participant showed that in seeds homozygous for recessive alleles of maize lpa1, steady state accumulations of the diphospho-inositol phosphates Ins P7 and Ins P8 are clearly evident in seeds that also have low levels of Ins P6. This interesting finding has opened up a new path for this research. Communication between cooperators on this project is via the internet and via e-mail communication.