A Reverse Genetics Approach to Characterizing Genes Involved in Seed Phytic Acid Biosynthesis and Accumulation.

A reverse genetics approach using Arabidopsis T-DNA insertion mutant lines has been employed to identify low phytic acid (lpa) genes and to examine the interaction and biological function of phytic acid metabolism genes. In addition to four genes previously implicated in seed phytic acid biosynthesis (AtIPK1, AtIPK2β, AtMRP5 and the Arabidopsis ortholog of rice OsLpa1), our analysis revealed that loss-of-function mutations in three other genes (AtITPK1, AtITPK4, and the Arabidopsis ortholog of maize lpa3) also resulted in lpa phenotypes. These genes represent possible targets for genetic engineering or mutation breeding of low phytate crops. Expression analysis of these genes revealed three distinct patterns in developing siliques which are consistent with their presumed roles. Enhancement and suppression of lpa gene expression in the mutants indicated that there is significant crosstalk between genes involved in seed phytic acid metabolism. Expression of Atipk1, which is involved in a late step in biosynthesis, and AtMRP4, which thought to be involved in transport/accumulation, were significantly enhanced in knockout mutants of AtIPK2β, AtITPK1 and AtITPK4 genes, which are involved in the intermediate steps of biosynthesis. In contrast, most lpa genes were suppressed in knockout mutants of AtMik, which encodes a myo-inositol kinase involved in an early step of phytic acid biosynthesis. All of the Arabidopsis lpa mutants were observed to be susceptible to salt and osmotic stress, suggesting an antioxidant function for phytic acid in seed physiology.