Perhaps the single most distinctive feature of plant inositol phosphate metabolism is the accumulation of inositol hexakisphosphate (InsP 6 ) 2 to levels up to several percent of dry weight in seed or storage tissues (Raboy and Dickinson, 1987) and in vegetative tissues to levels that are likely to be in excess of other inositol phosphates. It is likely that the inositol phosphates commonly found in plants are not related to the signaling molecule Ins(1,4,5) P 3 , but are intermediates of the pathways of InsP 6 synthesis and breakdown.We have described a number of inositol phosphates in the duckweed Spirodela polyrhiza (Brearley and Hanke, 1996a) and in barley aleurone tissue (Brearley and Hanke, 1996c). InsP 4 and InsP 5 species have been identified in mung bean (Stephens, 1990;Stephens et al., 1991) and soybean (Phillippy et al., 1994). Metabolic evidence (Brearley and Hanke, 1996b) suggests that some of those identified in S. polyrhiza are intermediates in InsP 6 biosynthesis. However, as there is some uncertainty surrounding the order of addition of the 1-and 5-Ps (fourth and fifth in the sequence proposed) to the inositol moiety of InsP 6 , the validity of the proposed order of the metabolic sequence relies heavily on the nature of the inositol phosphates identified, and, paradoxically, on those present but not yet described. The identification of enzyme activities that phosphorylate endogenous inositol phosphates to products higher in the sequence is of crucial importance, therefore, in distinguishing between possible pathways. Furthermore, as the pathway described in plants differs from that described in Dictyostelium discoideum (Stephens and Irvine, 1990) (notwithstanding a report of an alternative nuclear pathway in this organism; Van der Kaay et al., 1995), while that in animals is unclear, the nature of the pathway operating in plants assumes greater significance because it may shed light on the pathways operating in other kingdoms.Insofar as the patterns of isomers detected in plants are atypical of animal cells and may be indicative of functions for these compounds specific to plants, we have also set out to identify the range of InsP 3 species present in two experimental systems: frond tissue of the aquatic monocotyledonous plant S. polyrhiza (mesophyll cells predominantly) and root suspension cultures of the dicotyledonous plant Arabidopsis, which as a model experimental system in plant molecular genetics merits attention.
MATERIALS AND METHODS
Plant MaterialSpirodela polyrhiza L. plants were labeled with myo-[2-3 H]inositol (21 Ci/mmol, Amersham International, Buckinghamshire, UK) as described previously (Brearley and Hanke, 1996a). Root cell suspension cultures of Arabidopsis (ecotype Landsberg erecta) were obtained from Paul Duprée of the Department of Biochemistry, University of Cambridge. Stock cultures were maintained at 25°C on an orbital shaker in Gamborg's B5 medium (Sigma G-5893, Sigma Chemical, Poole, Dorset, UK) and further supple-
