Molecular and Biochemical Characterization of Two Plant Inositol Polyphosphate 6-/3-/5-Kinases

Despite the high deposition of inositol hexakisphosphate (IP6), also known as phytate or phytin, in certain plant tissues little is known at the molecular level about the pathway(s) involved in its production. In budding yeast, IP6 synthesis occurs through the sequential phosphorylation of I(1,4,5)P3 by two gene products, Ipk2 and Ipk1, a IP3/IP4 dual-specificity 6-/3-kinase and an inositol 1,3,4,5,6-pentakisphosphate 2-kinase, respectively. Here we report the identification and characterization of two inositol polyphosphate kinases from Arabidopsis thaliana, designated AtIpk2α and AtIpk2β that are encoded by distinct genes on chromosome 5 and that are ubiquitously expressed in mature tissue. The primary structures of AtIpk2α and AtIpk2β are 70% identical to each other and 12–18% identical to Ipk2s from yeast and mammals. Similar to yeast Ipk2, purified recombinant AtIpk2α and AtIpk2β have 6-/3-kinase activities that sequentially phosphorylate I(1,4,5)P3 to generate I(1,3,4,5,6)P5 predominantly via an I(1,4,5,6)P4 intermediate. While I(1,3,4,5)P4is a substrate for the plant Ipk2s, it does not appear to be a detectable product of the IP3 reaction. Additionally, we report that the plant and yeast Ipk2 have a novel 5-kinase activity toward I(1,3,4,6)P4 and I(1,2,3,4,6)P5, which would allow these proteins to participate in at least two proposed pathways in the synthesis of IP6. Heterologous expression of either plant isoform in an ipk2 mutant yeast strain restores IP4 and IP5 production in vivo and rescues its temperature-sensitive growth defects. Collectively our results provide a molecular basis for the synthesis of higher inositol polyphosphates in plants through multiple routes and indicate that the 6-/3-/5-kinase activities found in plant extracts may be encoded by the IPK2 gene class. Despite the high deposition of inositol hexakisphosphate (IP6), also known as phytate or phytin, in certain plant tissues little is known at the molecular level about the pathway(s) involved in its production. In budding yeast, IP6 synthesis occurs through the sequential phosphorylation of I(1,4,5)P3 by two gene products, Ipk2 and Ipk1, a IP3/IP4 dual-specificity 6-/3-kinase and an inositol 1,3,4,5,6-pentakisphosphate 2-kinase, respectively. Here we report the identification and characterization of two inositol polyphosphate kinases from Arabidopsis thaliana, designated AtIpk2α and AtIpk2β that are encoded by distinct genes on chromosome 5 and that are ubiquitously expressed in mature tissue. The primary structures of AtIpk2α and AtIpk2β are 70% identical to each other and 12–18% identical to Ipk2s from yeast and mammals. Similar to yeast Ipk2, purified recombinant AtIpk2α and AtIpk2β have 6-/3-kinase activities that sequentially phosphorylate I(1,4,5)P3 to generate I(1,3,4,5,6)P5 predominantly via an I(1,4,5,6)P4 intermediate. While I(1,3,4,5)P4is a substrate for the plant Ipk2s, it does not appear to be a detectable product of the IP3 reaction. Additionally, we report that the plant and yeast Ipk2 have a novel 5-kinase activity toward I(1,3,4,6)P4 and I(1,2,3,4,6)P5, which would allow these proteins to participate in at least two proposed pathways in the synthesis of IP6. Heterologous expression of either plant isoform in an ipk2 mutant yeast strain restores IP4 and IP5 production in vivo and rescues its temperature-sensitive growth defects. Collectively our results provide a molecular basis for the synthesis of higher inositol polyphosphates in plants through multiple routes and indicate that the 6-/3-/5-kinase activities found in plant extracts may be encoded by the IPK2 gene class. inositol 1,4,5-trisphosphate high performance liquid chromatography glutathione S-transferase inositol polyphosphate 5-phosphatase catalytically inactive kinase Phosphorylation events are a vital part of intracellular communication and enable specific responses to various stimuli. As an asymmetric cyclitol harboring six hydroxyls, myo-inositol is a scaffold that has the potential to transduce a wealth of information through the combinatorial addition of phosphates. One such intracellular messenger, inositol 1,4,5-trisphosphate (IP3),1 which is a product of phosphatidylinositol 4,5-bisphosphate hydrolysis by phospholipase C, has been extensively characterized for its ability to specifically stimulate calcium release from intracellular stores (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6188) Google Scholar). IP3 also serves as a precursor to the synthesis of over 15 water-soluble IPs including higher phosphorylated species such as inositol tetrakisphosphate (IP4), inositol pentakisphosphate (IP5), and inositol hexakisphosphate (IP6), which have recently been implicated as messengers regulating processes such as mRNA export, transcription, DNA repair, channel activity, and membrane trafficking (reviewed in Refs. 2Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (349) Google Scholarand 3Irvine R.V. Schell M.J. Nature Reviews. 2001; 2: 327-338Crossref Scopus (542) Google Scholar). In addition, IP6 has been suggested as a reservoir for the synthesis of the diphosphoryl class of inositol polyphosphates (referred to as PP-IPs and in some cases IP7 and IP8) the functions of which are currently under investigation (4Shears S.B. Biochim. Biophys. Acta. 1998; 1436: 49-67Crossref PubMed Scopus (153) Google Scholar, 5Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar). In plants, higher inositol polyphosphates, which are generally defined as IPs with four or more phosphates, likely share similar signaling roles as in other eukaryotes, but they also serve a specialized function in seeds as storage pools of inositol, phosphate, and mineral nutrients (6Raboy V. Gerbasi P. Subcell. Biochem. 1996; 26: 257-285Crossref PubMed Scopus (85) Google Scholar, 7Loewus F.A. Murthy P.P.N. Plant Science. 2000; 150: 1-19Crossref Scopus (521) Google Scholar). During seed development, IP6, also known as phytate, accumulates in storage protein bodies as mixed salts of various minerals such as iron, calcium, magnesium, potassium, manganese, and zinc (6Raboy V. Gerbasi P. Subcell. Biochem. 1996; 26: 257-285Crossref PubMed Scopus (85) Google Scholar, 8Raboy V. Trends Plant Sci. 2001; 6: 458-462Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). In this form phytate often represents more than 1% of the dry weight of seeds (9Raboy V. Morre D.J. Boss W.F. Loewus F.A. Inositol Metabolism in Plants. Wiley-Liss, Inc., 1990: 55-76Google Scholar). During germination, the phytate-containing storage protein bodies are disassembled, and the IP6 is rapidly hydrolyzed by phytases (6Raboy V. Gerbasi P. Subcell. Biochem. 1996; 26: 257-285Crossref PubMed Scopus (85) Google Scholar, 7Loewus F.A. Murthy P.P.N. Plant Science. 2000; 150: 1-19Crossref Scopus (521) Google Scholar) releasing stored nutrients to the developing seedling before the plant is capable of absorbing them from the immediate environment. Ironically, the very properties that make phytate beneficial to the plant cause it to be an antinutrient to the animal that consumes it. The high concentration of phytate in food grains and its ability to chelate mineral cations and resistance to animal digestion compromises mineral absorption. Undigested phytate in animal waste also contributes to eutrophication and environmental phosphorus pollution (8Raboy V. Trends Plant Sci. 2001; 6: 458-462Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). For these reasons, there is significant interest in altering phytate production in agriculturally significant plants. Currently, very little is known about the genetics of phytate production in plants through either lipid dependent or independent pathways (8Raboy V. Trends Plant Sci. 2001; 6: 458-462Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Biochemically, I(1,4,5)P3, I(1,3,4,5)P4, and I(1,3,4,5,6)P5 kinase activities have been described in pea roots and immature soybean seeds, respectively, (10Chattaway J.A. Drobak B.K. Watkins P.A.C. Dawson A.P. Letcher A.J. Stephens L.R. Irvine R.F. Planta. 1992; 187: 542-545Crossref PubMed Scopus (22) Google Scholar, 11Phillippy B.Q. Ullah A.H. Ehrlich K.C. J. Biol. Chem. 1994; 269: 28393-28399Abstract Full Text PDF PubMed Google Scholar, 12Phillippy B.Q. Plant Physiol. 1998; 116: 291-297Crossref Scopus (25) Google Scholar), but none of the corresponding genes have been identified. Several studies have indicated that IP6 may also be synthesized in lipid-independent pathways directly from inositol and/or inositol 3-phosphate (13Stephens L.R. Irvine R.F. Nature. 1990; 346: 580-583Crossref PubMed Scopus (131) Google Scholar, 14Brearley C.A. Hanke D.E. Biochem. J. 1996; 314: 227-233Crossref PubMed Scopus (88) Google Scholar). Despite this, only one IP kinase from any plant species has been cloned and thisArabidopsis gene product has I(1,3,4)P35/6-kinase activity (15Wilson M.P. Majerus P.W. Biochem. Biophys. Res. Commun. 1997; 232: 678-681Crossref PubMed Scopus (55) Google Scholar). Recently a pathway for IP6 synthesis has been delineated in the budding yeast Saccharomyces cerevisiae through a phospholipase C-dependent pathway in which IP3is sequentially phosphorylated to IP6 by the products of two genes, IPK2 and IPK1 (16York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar, 17Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar). Ipk2 is a dual-specificity IP3/IP4 6-/3-kinase that generates IP5 from IP3 (17Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar, 18Saiardi A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar), and Ipk1 is an I(1,3,4,5,6)P5 2-kinase (16York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar). When either of these two genes is dysfunctional in yeast, IP6 is no longer synthesized (16York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar, 17Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar). Here we report that the plant A. thaliana has two genes that encode IP3/IP4kinases, and characterization of their substrate specificities suggests that they have the potential to participate in multiple pathways of IP6 synthesis. in in the and to for by the A. J. PubMed Google in this in a The for the of and and by Arabidopsis synthesized as described Boss W.F. J. Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar), and the of by of each with the The and the The for and The for of and The and products respectively, and to from by the to the of the The with a in Biol. 1998; Full Text Full Text PDF PubMed Google Scholar) and with the for glutathione S-transferase in the expression a of by The the and by in with a yeast in with or by the yeast expression the Arabidopsis designated and and to various cerevisiae IP kinase in to generate a in the of IP in this have been to IP kinase activity of Ipk2 (17Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar). The for and to to the to be and identical to the for the in the it to an The as and the in the The for as and in with the to the described The high and the suggested by the independent for each products that purified and in a that only the that from the of the of the The of this a with a The product with and and and for that the the we expressed the AtIpk2α and AtIpk2β as proteins in and purified and them for I(1,4,5)P3 kinase activity as described for with as the of either of the purified recombinant proteins to phosphorylate I(1,4,5)P3 in not The each to One of with a of the and at to an of at by the addition of The for at by at and in of The at on and by 5 through a high for and to of and respectively. The to at for 15 at The with of a for 15 at with The with of and the proteins from the with of glutathione in and stored at in IPs from for which from IPs from for and which as phosphorylated by of recombinant in of and of inositol polyphosphate 5-phosphatase for at The by at for with of an IP4 2-kinase R. A. and J. in under the as described For of purified recombinant or with or for designated at in by the addition of the of and by the IP4 product of I(1,4,5)P3 phosphorylation or of and with as described for at that the of the product IP4 and not of the by the addition of of and the other with of purified recombinant inositol polyphosphate 5-phosphatase for at The and by over a and a from to over by for with IPs on to the of known IP AtIpk2α and AtIpk2β phosphorylated a of of recombinant AtIpk2α and AtIpk2β with I(1,4,5)P3, I(1,3,4,5)P4, or IP6 in and of for at The either by the addition of or to a concentration of and 5 and the at for by as described cerevisiae either or in of and and to The and the the IPs to York (16York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar). for from the and of A. thaliana plants to the the Plant with to any to the at for 5 with to for of of at for in a of of and of The by at for The of the of the and for each and The at for an and of DNA at for for and for The to the of and As a to for as described directly from the The for and The for and The to and The is a of inositol polyphosphate described as to IP3 and it by to be a of a protein IP3 known as Ipk2 or and diphosphoryl IP known as A. Caffrey J.J. Snyder S.H. Shears S.B. J. Biol. Chem. 2000; 275: 24686-24692Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 17Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar, M.J. Letcher A.J. C.A. J. Irvine R.F. FEBS Lett. 1999; PubMed Scopus Google Scholar, A. P. Snyder S.H. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. Snyder S.H. Sci. A. 2001; PubMed Scopus Google Scholar, Science. 1990; PubMed Scopus Google Scholar, V. PubMed Scopus Google Scholar). this is not found in IP kinases that phosphorylate the of the inositol (16York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar, E.B. J. Wente S.R. York J.D. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar), in (15Wilson M.P. Majerus P.W. Biochem. Biophys. Res. Commun. 1997; 232: 678-681Crossref PubMed Scopus (55) Google Scholar, M.P. Majerus P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). In to plant we the Arabidopsis for this the by the found two and harboring the and respectively. of the two IP kinases that they are 70% identical and similar to each other at the level of primary The of and are 12–18% identical and similar to cerevisiae Ipk2 and inositol polyphosphate which are characterized dual-specificity IP3/IP4 When the yeast, and are with other of the kinase that are The the and is the IP Biochem. J. 1997; PubMed Scopus Google Scholar). we the substrate of purified and expressed as The of IP kinases in this are by of the products two I(1,4,5)P3 and IP6. When either recombinant protein with and the product I(1,3,4,5,6)P5 through an IP4 indicated that the plant kinases the dual-specificity class harboring IP3/IP4 kinase activity to cerevisiae Ipk2 and When for kinase activity IP6 recombinant protein detectable diphosphoryl IP we have designated and as AtIpk2α and respectively. A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar, Biochem. J. PubMed Scopus Google Scholar) have that the and yeast Ipk2 generate as a we the of the IP4 in the plant IP3 to IP5 reaction. this, we the inositol polyphosphate which is known to the from and I(1,4,5)P3 but not AtIpk2α and AtIpk2β with IP3 and the IP4 of as in at which recombinant 5-phosphatase As in 5-phosphatase does not IP4 by either AtIpk2α or The 5-phosphatase in these it the I(1,4,5)P3 substrate to and in the it hydrolyzed to generate similar results this for yeast Ipk2 (16York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar). the Shears report of yeast Ipk2 A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar), we recombinant in these and found AtIpk2α and yeast Ipk2 I(1,4,5,6)P4 as the product of IP3 phosphorylation not substrate specificities have been described for of the IP kinase we a of IPs as The recombinant proteins for the ability to phosphorylate a of inositol species as described under activity I(1,4,5)P3, I(1,3,4,5)P4, As AtIpk2α and AtIpk2β phosphorylated I(1,4,5)P3 to IP4 and and with the addition of the with each phosphorylated the IP4 product to I(1,3,4,5,6)P5 and The plant IP kinases also phosphorylated I(1,4,5,6)P4 and as as and AtIpk2α and AtIpk2β to phosphorylate I(1,3,4,6)P4 and on the to generate I(1,3,4,5,6)P5 and IP6, our this 5-kinase activity has not been described for any of the IP kinase In these the kinases not phosphorylate inositol, or IP6 and AtIpk2β are inositol polyphosphate proteins of AtIpk2α and AtIpk2β with or for at products by and with the of known in the substrate specificities of AtIpk2α and AtIpk2β for I(1,4,5,6)P4 and the to phosphorylate than on the of from to to form IP5 as with the of the to I(1,4,5,6)P4 to form IP5 and under the to the of the recombinant for its various we the of found that AtIpk2α similar for I(1,4,5)P3 and of and respectively. are very similar to that for Ipk2 with a for I(1,4,5)P3 of Caffrey J.J. Shears S.B. FEBS Lett. 2001; PubMed Scopus Google Scholar). The of AtIpk2α for I(1,4,5,6)P4 that of IP3 and at The similar for phosphorylation of I(1,4,5,6)P4 and of I(1,4,5)P3 with a of of recombinant in a the plant gene products function as IP3/IP4 kinases in we in yeast Ipk2 As (17Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar) and in to with there a of IP production of The of this an of I(1,4,5)P3 and the of an the of of the I(1,4,5)P3 (17Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar). When or expressed from a yeast expression in these phosphorylation of IP3 and IP4 and the production of the IP6, as with a strain cerevisiae that Ipk2 activity have growth at as with and are to at (17Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar). the of IP production by AtIpk2α or AtIpk2β in mutant yeast the growth we expressed the plant kinases in the strain and in a strain with a catalytically inactive Ipk2 and the growth of these at When either or expressed in growth at The of growth at dependent on the IP kinase activity as indicated by the of the catalytically inactive of Ipk2, or AtIpk2β in the to growth at higher The Shears and Snyder have that the IP3/IP4 kinases are kinases that are to I(1,4,5)P3 to a of products that IP6, and/or A. P. Snyder S.H. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, A. A. Snyder S.H. Sci. A. 2001; PubMed Scopus Google Scholar, Caffrey J.J. Shears S.B. FEBS Lett. 2001; PubMed Scopus Google Scholar). As described in this we found that a novel activity that generates is not a kinase product of In to we the plant kinases yeast Ipk2 for that with I(1,4,5)P3 we IP4 and I(1,3,4,5,6)P5 products but not it is that we not have the plant kinases for these activities in through studies and mutant yeast Ipk1 and have been to be IP6 and respectively. As in the plant IP kinases are expressed in yeast on of the production of IP6. or not capable of IP3 to IP6 in we expressed the plant kinases in an When either or expressed in this mutant strain and the with the production of IP4 and IP5 but there no synthesis of IP6 identical to that of an strain (16York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar). the proteins in a recently characterized diphosphoryl that compromises production and to at Shears S.B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of either not production as through inositol expression the of the strain not In expression of diphosphoryl does production and the growth of mutant and J. in vivo provide that the plant Ipk2s described their yeast are IP3/IP4 kinases and not appear to have significant kinase activities as proposed by and appear to be we to the expression of each gene product in plant from from and we found that and expressed in tissues at least in mature plants, are not expressed in tissues and are either or have functions in IP production. has been in as a and as a IP3 is not on basis these our that these are we have and has for each gene AtIpk2α is and AtIpk2β is The of in plants is an of pathways for phytate synthesis have been proposed (8Raboy V. Trends Plant Sci. 2001; 6: 458-462Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). One is an inositol lipid-independent pathway that occurs through sequential phosphorylation of inositol or inositol through a of proposed on studies in and (13Stephens L.R. Irvine R.F. Nature. 1990; 346: 580-583Crossref PubMed Scopus (131) Google Scholar, 14Brearley C.A. Hanke D.E. Biochem. J. 1996; 314: 227-233Crossref PubMed Scopus (88) Google Scholar). is through a phospholipase or pathway that IP3 and phytate through multiple the pathway a has been proposed to I(1,4,5)P3 I(1,3,4,6)P4 I(1,3,4,5,6)P5 IP6 (reviewed in Refs. 2Majerus P.W. Annu. Rev. Biochem. 1992; 61: 225-250Crossref PubMed Scopus (349) Google S.B. Biochim. Biophys. Acta. 1998; 1436: 49-67Crossref PubMed Scopus (153) Google Scholar). a to phytate synthesis is to through the of a M.P. Majerus P. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). in the pathway has been I(1,4,5)P3 I(1,4,5,6)P4 I(1,3,4,5,6)P5 IP6 (16York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar). is from studies in budding yeast that have defined the molecular and basis for this pathway via the of inositol polyphosphate Ipk2 and Ipk1 (16York J.D. Odom A.R. Murphy R. Ives E.B. Wente S.R. Science. 1999; 285: 96-100Crossref PubMed Scopus (448) Google Scholar, 17Odom A.R. Stahlberg A. Wente S.R. York J.D. Science. 2000; 287: 2026-2029Crossref PubMed Scopus (346) Google Scholar). The and characterization of two of the from allow to to IP6 may be synthesized in plants. The of our is that IP6 is synthesized in a similar to budding yeast in which phospholipase I(1,4,5)P3, which is to I(1,3,4,5,6)P5 via either AtIpk2α or is by the that the plant kinases are to generate I(1,3,4,5,6)P5 from in or in as yeast of this of IP6 from a of in which we have cloned an Arabidopsis Ipk1 that I(1,3,4,5,6)P5 to IP6. R. A. and J. in In we also that the 6-/3-/5-kinase activities and the multiple substrate specificities of and gene products may indicate that these proteins also participate in the other routes of synthesis. For the plant kinases are with I(1,3,4,6)P4 each a novel 5-kinase activity that which the reaction. that the pathway from the of an (15Wilson M.P. Majerus P.W. Biochem. Biophys. Res. Commun. 1997; 232: 678-681Crossref PubMed Scopus (55) Google Scholar) and an inositol polyphosphate 5-phosphatase J. Plant Physiol. 2001; PubMed Scopus Google Scholar). is not is would be in that there does not appear to be a specific I(1,4,5)P3 gene as is the of the defined by the not significant IP3 activity in the Shears and have A. Caffrey J.J. Snyder S.H. Shears S.B. FEBS Lett. 2000; 468: 28-32Crossref PubMed Scopus (119) Google Scholar, Biochem. J. PubMed Scopus Google Scholar, Caffrey J.J. Shears S.B. FEBS Lett. 2001; PubMed Scopus Google Scholar) that other Ipk2s as an of the IP3 to IP5 reaction. the described in this we have not been to as a product but it is that under would generate I(1,3,4,5)P4, which be by a 5-phosphatase to make there a the for lipid-independent and synthesis we have not been to directly protein for kinase activity toward of the proposed IP of the lipid-independent which and they are not as have and not pathways are in their of IP5 to a 2-kinase, Ipk1, which we have cloned from plants and from M.P. Majerus P.W. Wente S.R. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google is also that the activity Shears S.B. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), that this may be the kinase for the of to I(1,3,4,5,6)P5 the lipid-independent that to pathways is the synthesis of Several have that a of plants inositol 3-phosphate similar in activity to the yeast gene 3-phosphate this is described in the as The occurs through inositol that generate inositol from which have also been found to be from yeast to that and pathways of IP6 synthesis would pathway under of inositol or such as that seed the of is not to which synthesis pathway is seed provide molecular for synthesis through does not that plants the gene products to the of IP6 found in of function of or identification of the in certain phytate mutant plants distinct pathways to IP6 production to allow for of signaling and of phospholipase pathways generate I(1,4,5)P3 that is phosphorylated by certain of Ipk2s and to higher IPs involved in signaling pathways such as in the the seed is nutrients it may a pathway to such as to and to IP6 through phosphorylation The IP of each pathway are distinct The of multiple of Ipk2 described and are with in which the plant either through or the pathways to specifically in studies of the plant gene products involved in these pathways likely the routes of synthesis and enable the specific of pathways the plant for and Shears for the recombinant