Inositol phosphates from barley low-phytate grain mutants analysed by metal-dye detection HPLC and NMR

Inositolphosphates from barley low-phytate grain mutants and their parent v ariety were analysed by metal-dye detection HPLC and NMR. Compound assignme nt was carried out by comparison of retention times using a chemical hydrol ysate of phytate [Ins(1,2,3,4,5,6)P-6] as a reference; Co-inciding retentio n times indicated the presence of phytate, D/L-Ins(1,2,3,4,5)P-5, Ins(1,2,3 ,4,6)P-6, D/L-(1,2,4,5,6)P-5, D/L-(1,2,3,4)P-4, D/L-Ins(1,2,5,6)P-4 and D/L -Ins(1,4,5,6)P-4 in PLP1B mutants as well as the parent variety. In grain e xtracts from mutant lines PLP1A, PLP2A and PLP3A unusual accumulations of D /L-Ins(1,3,4,5)P-4 were observed whereas phytate and the above-mentioned in ositol phosphates were present in relatively small amounts. Assignment of D /L-Ins(1,3,4,5)P-4 was corroborated by precise co-chromatography with a com mercial Ins(1,3,4,5)P-4 standard and by NMR spectroscopy. Analysis of inosi tol phosphates during grain development revealed accumulation of phytate an d D/L-Ins(1,3,4,5)P-4, which suggested the tetrakisphosphate compound to be an intermediate of phytate synthesis. This assumption was strengthened fur ther by phytate degradation assays showing that D/L-Ins(1,3,4,5)P-4 did not belong to the spectrum of degradation products generated by endogenous phy tase activity. Metabolic scenarios leading to accumulation of D/L-Ins(1,3,4 ,5)P-4 in barley low-phytate mutants are discussed.