Inositol phosphates from barley low-phytate grain mutants analysed by metal-dye detection HPLC and NMR.
Inositol phosphates from barley low-phytate grain mutants and their parent variety were analysed by metal-dye detection HPLC and NMR. Compound assignment was carried out by comparison of retention times using a chemical hydrolysate of phytate [Ins(1,2,3,4,5,6)P(6)] as a reference. Co-inciding retention times indicated the presence of phytate, D/L-Ins(1,2,3,4,5)P(5), Ins(1,2,3,4,6)P(5), 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 extracts 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 inositol 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 commercial Ins(1,3,4,5)P(4) standard and by NMR spectroscopy. Analysis of inositol phosphates during grain development revealed accumulation of phytate and 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 further 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 phytase activity. Metabolic scenarios leading to accumulation of D/L-Ins(1,3,4,5)P(4) in barley low-phytate mutants are discussed.
Hatzack
F
F
Plant Products and Biomass Recycling Programme, Plant Biology and Biogeochemistry Department, PBK-301, Risø National Laboratory, P.O. Box 49, DK-4000 Roskilde, Denmark.
Hübel
F
F
Zhang
W
W
Hansen
P E
PE
Rasmussen
S K
SK
D010832:6-Phytase / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D004396:Coloring Agents; D004986:Ether; D001467:Hordeum / Q000737:chemistry* / Q000235:genetics; D007295:Inositol Phosphates / Q000302:isolation & purification* / Q000378:metabolism*; D007536:Isomerism; D009682:Magnetic Resonance Spectroscopy; D008670:Metals; D010833:Phytic Acid / Q000032:analysis*; D014238:Trichloroacetic Acid
D004396:Coloring Agents; D007295:Inositol Phosphates; D008670:Metals; D004986:Ether; C081655:inositol-1,3,4,5-tetrakisphosphate; D014238:Trichloroacetic Acid; D010833:Phytic Acid; D010832:6-Phytase