Identification of genes necessary for wild-type levels of seed phytic acid in Arabidopsis thaliana using a reverse genetics approach

The majority of phosphorus (P) in seeds is found in phytic acid (InsP(6)) which accumulates as the mixed salt phytate. InsP(6) is generally considered to be an anti-nutrient and the development of low phytic acid (lpa) seed crops is of significant interest. We have employed a reverse genetics approach to examine the impact of disrupting genes involved in inositol phosphate metabolism on Arabidopsis seed InsP(6) levels. Our analysis revealed that knockout mutations in three genes (AtITPK1, AtITPK4, and AtMIK/At5g58730) reduced seed InsP(6) in addition to knockouts of four previously reported genes (AtIPK1, AtIPK2 beta, AtMRP5, and At5g60760). Seeds of these lpa mutants also exhibited reduced germination under various stress conditions. The greatest reduction in InsP(6) (>70%) was observed in atmrp5 seeds which were also among the least sensitive to the stresses examined. Expression analysis of the lpa genes revealed three distinct patterns in developing siliques consistent with their presumed roles. Disruption of each lpa gene resulted in changes in the expression in some of the other lpa genes indicating that transcription of lpa genes is modulated by other constituents of InsP(6) metabolism. While all the lpa genes represent possible targets for genetic engineering of low phytate seed crops, mutations in AtMRP5, AtMIK, and At5g60760 may be most successful for conventional approaches such as mutation breeding.