METABOLIC EVIDENCE FOR THE ORDER OF ADDITION OF INDIVIDUAL PHOSPHATE ESTERS TO THE MYOINOSITOL MOIETY OF INOSITOL HEXAKISPHOSPHATE IN THE DUCKWEED SPIRODELA-POLYRHIZA L

The aquatic monocotyledonous plant Spirodela polyrhiza was labelled wi th [P-32]P-i for short periods under non-equilibrium conditions. An In sP(6) fraction was obtained and dissected by using enantiospecific (en zymic) and non-enantiospecific (chemical) means to determine the relat ive labelling of individual phosphate substituents on the inositol rin g of InsP(6). Phosphates in positions D-1, -2, -3, -4, -5 and -6 conta ined approx. 21%, 32-39%, 9-10%, 14-16%, 19-23% and 16-18% of the labe l respectively. We conclude from the foregoing, together with identiti es [described in the preceding paper, Brearley and Hanke (1996) Bioche m. J. 314, 215-225] of inositol phosphates found in this plant at a de velopmental stage associated with massive accumulation of InsP(6), tha t synthesis of InsP(6) from myo-inositol proceeds according to the seq uence Ins3P --> Ins(3,4)P-2 --> Ins(3,3,6)P-3 --> Ins(3,4,5,6)P-4 --> Ins(1,3,4,5,6)P-5 --> InsP(6) in Spirodela polyrhiza. These results re present the first description of the synthetic sequence to InsP(6) in the plant kingdom and the only comprehensive description of endogenous inositol phosphates in any plant tissue. The sequence described diffe rs from that reported in the slime mould Dictyostelium discoideum.