Effect of atropine and gammahydroxybutyrate on inschemically induced changes in the level of radioactivity in [3H]inositol phosphates in gerbil brain in vivo

Brain ischemia in gerbils was induced by ligation of both common carotid arteries for 1 min or 10 min. Sham-operated animals served as controls. Intracerebral injection of [³H]inositol into gerbil brain 16 hr before ischemic insult resulted in equilibration of the label between inositol lipids and water-soluble inositol phosphate.A short ischemic period (1 min) resulted in a statistically significant increase in the radioactivity of inositol triphosphate (IP₃) and inositol monophosphate (IP), by about 48% and 79%, respectively, with little change in that of the intermediate inositol biphosphate (IP₂), which increased by about 16%. When the ischemic period was prolonged (10 min), an increase in the radioactivity of inositol monophosphate exclusively, by about 84%, was observed. The level of radioactivity in inositol phosphates IP₂ and IP₃ decreased by about 50%, probably as a consequence of phosphatase activation by the ischemic insult.The agonist of the cholinergic receptor, carbachol, injected intracerebrally (40 g per animal) increased accumulation of radioactivity in all inositol phosphates. The level of radioactivity in IP₃, IP₂, and IP was elevated by about 40, 23, and 147%, respectively.The muscarinic cholinergic antagonist, atropine, injected intraperitoneally in doses of 100 mg/kg body wt. depressed phosphoinositide metabolism in control animals. The level of radioactivity in water-soluble inositol metabolites in the brain of animals pretreated with atropine was evidently about 32% lower than in untreated animals.Pretreatment with atropine decreased the radioactivity of all inositol phosphates in the brain of animals subjected to 1-min ischemia and the radioactivity of IP in the case of 10-min brain ischemia. Gammabutyrolactone (GBL) administered intraperitoneally in the anesthetic dose 300 mg/kg body wt. diminished inositol monophosphate accumulation induced by either ischemic condition.Results from these in vivo studies are evidence that the blockage of cholinergic receptors by atropine depresses the response of phosphoinositides to physiological and particularly pathological stimuli.The results suggest that stimulation of the cholinergic receptor system is involved in the degradation of polyphosphoinositides during ischemia.     

Atrial natriuretic peptide inhibits the phosphoinositide hydrolysis in murine Leydig tumor cells

The ability of ANP to inhibit the hydrolysis of phosphoinositides was examined in [³H] myoinositol-labeled intact murine Leydig tumor (MA-10) cells. Arginine vasopressin (AVP) stimulated the formation of inositol monophosphate (IP₁), inositol bisphosphate (IP₂), and inositol trisphosphate (IP₃) both in a time- and dose- dependent manner in MA-10 cells. ANP inhibited the AVP-induced formation of IP₁, IP₂, and IP₃ in these cells. The inhibitory effect of ANP on the AVP-stimulated formation of IP₁, IP₂, and IP₃ accounted for 30%, 38% and 42%, respectively, which was observed at the varying concentrations of AVP. ANP caused a dose-dependent attenuation in AVP-stimulated production of IP₁, IP₂ and IP₃ with maximum inhibition at 100 nM concentration of ANP. The production of inositol phosphates was inhibited in the presence of 8- bromo cGMP in a dose-dependent manner, whereas dibutyryl-cAMP had no effect on the generation of these metabolites. The LY 83583, an inhibitor of guanylyl cyclase and cGMP production, abolished the inhibitory effect of ANP on the AVP-stimulated production of inositol phosphates. Furthermore, 10 M LY 83583 also inhibited the ANP-stimulated guanylyl cyclase activity and the intracellular accumulation of cGMP by more than 65–70%. The inhibition of eGMP-dependent protein kinase by H-8, significantly restored the levels of AVP-stimulated inositol phosphates in the presence of either ANP or exogenous 8-bromo cGMP. The results of this study suggest that ANP exerts an inhibitory effect on the production of inositol phosphates in murine Leydig tumor (MA-10) cells by mechanisms involving cGMP and cGMP-dependent protein kinase.Established Investigator of the American Heart Association     

Characterization of Inositol Phosphates in Carrot (Daucus carota L.) Cells

We have shown previously that inositol-1,4,5-trisphosphate (IP₃) stimulates an efflux of ⁴⁵Ca²⁺ from fusogenic carrot protoplasts (M Rincón, WF Boss [1987] Plant Physiol 83: 395-398). In light of these results, we suggested that IP₃ might serve as a second messenger for the mobilization of intracellular Ca²⁺ in higher plant cells. To determine whether or not IP₃ and other inositol phosphates were present in the carrot cells, the cells were labeled with myo-[2-³H]inositol for 18 hours and extracted with ice-cold 10% trichloroacetic acid. The inositol metabolites were separated by anion exchange chromatography and by paper electrophoresis. We found that [³H]inositol metabolites coeluted with inositol bisphosphate (IP₂) and IP₃ when separated by anion exchange chromatography. However, we could not detect IP₂ or IP₃ when the inositol metabolites were analyzed by paper electrophoresis even though the polyphosphoinositides, which are the source of IP₂ and IP₃, were present in these cells. Thus, [³H] inositol metabolites other than IP₂ and IP₃ had coeluted on the anion exchange columns. The data indicate that either IP₃ is rapidly metabolized or that it is not present at a detectable level in the carrot cells.     

Inositol polyphosphates contribute to cellular circadian rhythms: Implications for understanding lithium's molecular mechanism

Most living organisms maintain cell autonomous circadian clocks that synchronize critical biological functions with daily environmental cycles. In mammals, the circadian clock is regulated by inputs from signaling pathways including glycogen synthase kinase 3 (GSK3). The drug lithium has actions on GSK3, and also on inositol metabolism. While it is suspected that lithium's inhibition of GSK3 causes rhythm changes, it is not known if inositol polyphosphates can also affect the circadian clock. We examined whether the signaling molecule inositol hexaphosphate (IP₆) has effects on circadian rhythms. Using a bioluminescent reporter (Per2::luc) to measure circadian rhythms, we determined that IP₆ increased rhythm amplitude and shortened period in NIH3T3 cells. The IP₆ effect on amplitude was attenuated by selective siRNA knockdown of GSK3B and pharmacological blockade of AKT kinase. However, unlike lithium, IP₆ did not induce serine-9 phosphorylation of GSK3B. The synthesis of IP₆ involves the enzymes inositol polyphosphate multikinase (IPMK) and inositol pentakisphosphate 2-kinase (IPPK). Knockdown of Ippk had effects opposite to those of IP₆, decreasing rhythm amplitude and lengthening period. Ipmk knockdown had few effects on rhythm alone, but attenuated the effects of lithium on rhythms. However, lithium did not change the intracellular content of IP₆ in NIH3T3 cells or neurons. Pharmacological inhibition of the IP₆ kinases (IP6K) increased rhythm amplitude and shortened period, suggesting secondary effects of inositol pyrophosphates may underlie the period shortening effect, but not the amplitude increasing effect of IP₆. Overall, we conclude that inositol phosphates, in particular IP₆ have effects on circadian rhythms. Manipulations affecting IP₆ and related inositol phosphates may offer a novel means through which circadian rhythms can be regulated.     

IP3 signalling regulates exogenous RNAi in Caenorhabditis elegans

RNA interference (RNAi) is a widespread and widely exploited phenomenon. Here, we show that changing inositol 1,4,5‐trisphosphate (IP₃) signalling alters RNAi sensitivity in Caenorhabditis elegans. Reducing IP₃ signalling enhances sensitivity to RNAi in a broad range of genes and tissues. Conversely up‐regulating IP₃ signalling decreases sensitivity. Tissue‐specific rescue experiments suggest IP₃ functions in the intestine. We also exploit IP₃ signalling mutants to further enhance the sensitivity of RNAi hypersensitive strains. These results demonstrate that conserved cell signalling pathways can modify RNAi responses, implying that RNAi responses may be influenced by an animal's physiology or environment.     

KCS1 deletion in Saccharomyces cerevisiae leads to a defect in translocation of autophagic proteins and reduces autophagosome formation

Inositol phosphates are implicated in the regulation of autophagy; however, the exact role of each inositol phosphate species is unclear. In this study, we systematically analyzed the highly conserved inositol polyphosphate synthesis pathway in S. cerevisiae for its role in regulating autophagy. Using yeast mutants that harbored a deletion in each of the genes within the inositol polyphosphate synthesis pathway, we found that deletion of KCS1, and to a lesser degree IPK2, led to a defect in autophagy. KCS1 encodes an inositol hexakisphosphate/heptakisposphate kinase that synthesizes 5-IP₇ and IP₈; and IPK2 encodes an inositol polyphosphate multikinase required for synthesis of IP₄ and IP₅. We characterized the kcs1Δ mutant strain in detail. The kcs1Δ yeast exhibited reduced autophagic flux, which might be caused by both the reduction in autophagosome number and autophagosome size as observed under nitrogen starvation. The autophagy defect in kcs1Δ strain was associated with mislocalization of the phagophore assembly site (PAS) and a defect in Atg18 release from the vacuole membrane under nitrogen deprivation conditions. Interestingly, formation of autophagosome-like vesicles was commonly observed to originate from the plasma membrane in the kcs1Δ strain. Our results indicate that lack of KCS1 interferes with proper localization of the PAS, leads to reduction of autophagosome formation, and causes the formation of autophagosome-like structure in abnormal subcellular locations.     

Analysis of Dictyostelium discoideum Inositol Pyrophosphate Metabolism by Gel Electrophoresis

The social amoeba Dictyostelium discoideum was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study in vitro enzymatic reactions. Here we employ PAGE technology to characterize the D. discoideum inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP₆ or Phytic acid) and its derivative inositol pyrophosphates, IP₇ and IP₈. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP₉ in D. discoideum cells, a molecule so far detected only from in vitro biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP₅) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in D. discoideum in the vegetative state than previously detected. A three-fold increase in IP₈ was observed during development of D. discoideum a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP₅K, doesn’t’ play a major role in the IP₈ developmental increase.     

Inositol 1,4,5‐trisphosphate receptor regulates replication,differentiation, infectivity and virulence of the parasitic protist Trypanosoma cruzi

In animals, inositol 1,4,5‐trisphosphate receptors (IP₃Rs) are ion channels that play a pivotal role in many biological processes by mediating Ca²⁺ release from the endoplasmic reticulum. Here, we report the identification and characterization of a novel IP₃R in the parasitic protist, Trypanosoma cruzi, the pathogen responsible for Chagas disease. DT40 cells lacking endogenous IP₃R genes expressing T. cruzi IP₃R (TcIP₃R) exhibited IP₃‐mediated Ca²⁺ release from the ER, and demonstrated receptor binding to IP₃. TcIP₃R was expressed throughout the parasite life cycle but the expression level was much lower in bloodstream trypomastigotes than in intracellular amastigotes or epimastigotes. Disruption of two of the three TcIP₃R gene loci led to the death of the parasite, suggesting that IP₃R is essential for T. cruzi. Parasites expressing reduced or increased levels of TcIP₃R displayed defects in growth, transformation and infectivity, indicating that TcIP₃R is an important regulator of the parasite's life cycle. Furthermore, mice infected with T. cruzi expressing reduced levels of TcIP₃R exhibited a reduction of disease symptoms, indicating that TcIP₃R is an important virulence factor. Combined with the fact that the primary structure of TcIP₃R has low similarity to that of mammalian IP₃Rs, TcIP₃R is a promising drug target for Chagas disease.     

KRAS-induced actin-interacting protein is required for the proper localization of inositol 1,4,5-trisphosphate receptor in the epithelial cells

Three inositol 1,4,5-trisphosphate receptor (IP₃R) subtypes are differentially expressed among tissues and function as the Ca²⁺ release channel on specialized endoplasmic reticulum (ER) membranes. The proper subcellular localization of IP₃R is crucial for its proper function, but this molecular mechanism is unclear. KRAS-induced actin-interacting protein (KRAP) was originally identified as a cancer-related molecule, and is involved in the regulation of whole-body energy homeostasis and pancreatic exocrine system. We herein identified IP₃R as an associated molecule with KRAP in vivo, and the association was validated by the co-immunoprecipitation and confocal immunostaining studies in mouse tissues including liver and pancreas. The association of KRAP with IP₃R was also observed in the human epithelial cell lines including HCT116, HeLa and HEK293 cells. Intriguingly, KRAP interacts with distinct subtypes of IP₃R in a tissue-dependent manner, i.e. IP₃R1 and IP₃R2 in the liver and IP₃R2 and IP₃R3 in the pancreas. The NH₂-terminal amino acid residues 1–610 of IP₃R are critical for the association with KRAP and KRAP–IP₃R complex resides in a specialized ER but not a typical reticular ER. Furthermore, the localization of particular IP₃R subtypes in tissues from KRAP-deficient mice is obviously disturbed, i.e. IP₃R1 and IP₃R2 in the liver and IP₃R2 and IP₃R3 in the pancreas. These findings demonstrate that KRAP physically associates with IP₃R and regulates the proper localization of IP₃R in the epithelial cells in vivo and cultured cells, and might shed light on the Ca²⁺ signaling underlying physiological cellular programs, cancer development and metabolism-related diseases.     

The function of inositol high polyphosphate binding proteins

The inositol phosphate metabolism network has been found to be much more complex than previously thought, as more and more inositol phosphates and their metabolizing enzymes have been discovered. Some of the inositol phosphates have been shown to have biological activities, but little is known about their signal transduction mechanisms except for that of inositol 1,4,5-trisphosphate. The recent discovery, however, of a number of binding proteins for inositol high polyphosphate [inositol 1,3,4,5-tetrakisphosphate (IP₄), inositol 1,3,4,5,6-pentakisphosphate, or inositol hexakisphosphate] enables us to speculate on the physiological function of these compounds. In this article we focus on two major issues: (1) the roles of inositol high polyphosphates in vesicular trafficking, especially exocytosis, and (2) pleckstrin homology domaincontaining IP4 binding proteins involved in the Ras signaling pathway.     

Grainyhead-like 2 regulates neural tube closure and adhesion molecule expression during neural fold fusion

Defects in closure of embryonic tissues such as the neural tube, body wall, face and eye lead to severe birth defects. Cell adhesion is hypothesized to contribute to closure of the neural tube and body wall; however, potential molecular regulators of this process have not been identified. Here we identify an ENU-induced mutation in mice that reveals a molecular pathway of embryonic closure. Line2F homozygous mutant embryos fail to close the neural tube, body wall, face, and optic fissure, and they also display defects in lung and heart development. Using a new technology of genomic sequence capture and high-throughput sequencing of a 2.5 Mb region of the mouse genome, we discovered a mutation in the grainyhead-like 2 gene (Grhl2). Microarray analysis revealed Grhl2 affects the expression of a battery of genes involved in cell adhesion and E-cadherin protein is drastically reduced in tissues that require Grhl2 function. The tissue closure defects in Grhl2 mutants are similar to that of AP-2α null mutants and AP-2α has been shown to bind to the promoter of E-cadherin. Therefore, we tested for a possible interaction between these genes. However, we find that Grhl2 and AP-2α do not regulate each other's expression, E-cadherin expression is normal in AP-2α mutants during neural tube closure, and Grhl2;AP-2α trans-heterozygous embryos are morphologically normal. Taken together, our studies point to a complex regulation of neural tube fusion and highlight the importance of comparisons between these two models to understand more fully the molecular pathways of embryonic tissue closure.     

Intracellular signalling of epinephrine in rat hepatocytes during fetal development and hepatic regeneration

The changes in intracellular calcium concentration and IP₃ production after the addition of epinephrine were analysed in adult, fetal (20th–22nd day of intrauterine life), and regenerating rat hepatocytes (4 h–24 h after partial hepatectomy) to determine whether the signal transduction is the same in quiescent proliferating and differentiating cells.The epinephrine treatment causes a significative cytosolic calcium transient in hepatocytes isolated in the last day of fetal life (22-day old) and in the early stage of regeneration (4 h). This effect is not significant in the previous stage of fetal life (20-day old) and at the onset of M phase of cell cycle after partial hepatectomy (24 h).[³H]myo inositol incorporation into IP₃ and IP₄ is higher in 20 day fetal and regenerating hepatocytes with respect to the control. In these cells the epinephrine does not affect basal level of IP₃ and IP₄, while it causes a substantial increase of these inositol phosphates in adult hepatocytes.[³H]myo inositol incorporation into PIP₂ is very low at the 20th day of fetal life. Epinephrine has no effect on this parameter in fetal and regenerating hepatocytes.Our results show that the epinephrine signal is mediated differently in proliferating and in quiescent hepatocytes.     

The inositol 1,4,5‐trisphosphate receptor is required for maintenance of olfactory adaptation in Drosophila antennae

A role for inositol 1,4,5‐trisphosphate (IP₃) as a second messenger during olfactory transduction has been postulated in both vertebrates and invertebrates. However, given the absence of either suitable pharmacological reagents or mutant alleles specific for the IP₃ signaling pathway, an unequivocal demonstration of IP₃ function in olfaction has not been possible. Here we have investigated the role of a well‐established cellular target of IP₃—the IP₃ receptor (IP₃R)—in olfactory transduction in Drosophila. For this purpose we tested existing viable combinations of IP₃R mutant alleles, as well as a newly generated set of viable itpr alleles, for olfactory function. In all of the viable allelic combinations primary olfactory responses were found to be normal. However, a subset of itpr alleles (including a null allele) exhibit faster recovery after a strong pulse of odor, indicating that the IP₃R is required for maintenance of olfactory adaptation. Interestingly, this defect in adaptation is dominant for two of the alleles tested, suggesting that the mechanism of adaptation is sensitive to levels of the IP₃R. © 2000 John Wiley & Sons, Inc. J Neurobiol 43: 282–288, 2000     

Differential expression of type 2 and type 3 inositol 1,4,5-trisphosphate receptor mRNAs in various mouse tissues: in situ hybridization study

The inositol 1,4,5-trisphosphate receptor (IP₃R) is an intracellular Ca²⁺ release channel responsible for mobilizing stored Ca²⁺. Three different receptor types have been molecularly cloned, and their genes have been classified into a family. The gene for the type 1 receptor (IP₃R1) is predominantly expressed in cerebellar Purkinje neurons, but its gene product is localized widely in a variety of tissues; however, there is little information on what types of cells express the other two receptor types, type 2 and type 3 (IP₃R2 and IP₃R3, respectively). We studied the expression of the IP₃R gene family in various mouse tissues by in situ hybridization histochemistry. Compared with IP₃R1, the levels of expression of IP₃R2 and IP₃R3 mRNAs were low in all of the tissues tested. IP₃R2 mRNA was localized in the intralobular duct cells of the submandibular gland, the urinary tubule cells of the kidney, the epithelial cells of epididymal ducts and the follicular granulosa cells of the ovary, while the IP₃R3 mRNA was distributed in gastric cells, salivary and pancreatic acinar cells and the epithelium of the small intestine. All of these cells which express either IP₃R2 or IP₃R3 mRNA are known to have a secretory function in which IP₃/Ca²⁺ signalling has been shown to be involved, and thus either IP₃R2 or IP₃R3 may be a prerequisite to secretion in these cells.     

Roles for inositol polyphosphate kinases in the regulation of nuclear processes and developmental biology

Our laboratory studies the biology and enzyme regulation of inositol signal transduction pathways, which are activated in response to a wide range of stimuli. As a six-carbon cyclitol, inositol and its numerous phosphorylated derivatives efficiently generate combinatorial ensembles of signaling molecules. Through the cloning and characterization of inositol polyphosphate kinases (IPK), novel roles for inositol tetrakisphosphate (IP₄), inositol pentakisphosphate (IP₅), and inositol hexakisphosphate (IP₆) and inositol pyrophosphates (PP-IPs), have been identified. Studies have linked the IPKs and their inositide products to the regulation of nuclear processes including gene expression, chromatin remodeling, mRNA export, DNA repair and telomere maintenance. Analysis of IPK knockout animals has revealed a role for production of IPs in regulation of embryogenesis and organism development.The discoveries of the IPK proteins and their connection to nuclear signaling have generated significant interest in the field. Furthermore, they have provided interesting clues into the evolution of inositide-signaling pathways. Ipk2/IPMK and IPS/IP6K family members are conserved from yeast to man. In contrast, the IP₃ 3-kinase (ITPK) branch is observed in selected metazoans and not in plant or fungi. This may imply that Ipk2 and IPS activities evolved first among the group. The promiscuity of the Ipk2 protein further supports this notion and may provide the cell with a means to generate many IP species in a genetically economical fashion. Studies of yeast inositide signaling reveal that these simple eukaryotes do not have an IP₃ receptor in their genome and do not utilize diacylglycerol to activate protein kinase C. Thus, it appears that the canonical “text book” aspects of inositide-signaling pathways are not conserved throughout eukaryotic evolution. In light of the conservation of Ipk2/IPMK, Ipk1 and IPS/IP6K pathways from yeast to man it is interesting to speculate that a primordial role of phospholipase C-induced, IPK-dependent inositide signaling was to regulate nuclear processes. As calcium and PKC signaling evolved in metazoans, these may have greatly enhanced signaling capabilities. Recent studies demonstrating an essential role for IP₅, IP₆ and possibly PP-IP production in metazoan development highlight the importance of IPK signaling in cellular responses in metazoans. With these thoughts in mind, we eagerly await future studies aimed at further elucidating how these signaling codes participate in developmental processes and the control of gene expression, mRNA export, and DNA metabolism.     

A unique missense allele of BAF155, a core BAF chromatin remodeling complex protein,causes neural tube closure defects in mice

Failure of embryonic neural tube closure results in the second most common class of birth defects known as neural tube defects (NTDs). While NTDs are likely the result of complex multigenic dysfunction, it is not known whether polymorphisms in epigenetic regulators may be risk factors for NTDs. Here we characterized Baf155^msp3, a unique ENU‐induced allele in mice. Homozygous Baf155^mps3 embryos exhibit highly penetrant exencephaly, allowing us to investigate the roles of an assembled, but malfunctional BAF chromatin remodeling complex in vivo at the time of neural tube closure. Evidence of defects in proliferation and apoptosis were found within the neural tube. RNA‐Seq analysis revealed that surprisingly few genes showed altered expression in Baf155 mutant neural tissue, given the broad epigenetic role of the BAF complex, but included genes involved in neural development and cell survival. Moreover, gene expression changes between individual mutants were variable even though the NTD was consistently observed. This suggests that inconsistent gene regulation contributes to failed neural tube closure. These results shed light on the role of the BAF complex in the process of neural tube closure and highlight the importance of studying missense alleles to understand epigenetic regulation during critical phases of development. © 2013 Wiley Periodicals, Inc. Develop Neurobiol 74: 483–497, 2014     

Phosphate mobilization in grains of Hordeum distichon

When decorticated grains were germinated at 14·5°, inorganic substances moved from the endosperm, mainly the aleurone layer, to the embryo. The level of Pi rose in the embryo and endosperm, and the embryo appeared to accumulate Pi against a concentration gradient. The level of organic-P declined in the endosperm, particularly in the alcurone layer. Separated aleurone layers, incubated at 25°, released only small amounts of organic or inorganic phosphate. However, when incubated with gibberellic acid (GA₃), a massive release of Pi occurred at the expense of organic phosphates within the tissue. This release followed a sigmoid pattern with time following a lag and was virtually complete in 6 days. Allowing the aleurone layer to dry before incubating with GA₃ reduced or abolished the lag period of Pi release and only marginally depressed the total amount ultimately freed. In contrast, α-amylase production was depressed by the longer periods of drying. The major phosphate of the aleurone was phytate (meso-inositol hexaphosphate, IP₆), but traces of 1P₄, IP₃, IP₂, IP₁, Pi and unidentified phosphates were detected. During incubation with GA₃ the IP₆ content fell, and the lower esters of inositol rose slightly and then fell in a pattern indicating that the phosphate groups of each IP₆ molecule were being sequentially hydrolysed. After 6 days incubation, the tissue phosphates were reduced to a very low level. Attempts to isolate aleurone grains containing phytate were unsuccessful.