Differentially Expressed Forms of 1-L- -Inositol-1-Phosphate Synthase (EC) in

Abstract

We have characterized two distinct polypeptides with 1-L-myo-inositol-1-phosphate synthase (MI-1-P synthase) activity that are differentially expressed during development in Phaseolus vulgaris. Western analyses, enzyme assays, and partial purification of MI-1-P synthase during embryonic and postembryonic development show that its expression is temporally and spatially regulated.Developmental Western analyses of soluble proteins detect a small protein, approximately 33 kDa, with MI-1-P synthase activity during the globular stage (stage II) of embryogenesis and in mature roots. Expression of this small protein is also enriched in thylakoidal membranes of fractionated leaf chloroplasts, although Western analyses of total soluble leaf proteins show no cross-reacting material. In contrast, a larger protein, approximately 56 kDa, with MI-1-P synthase activity is present during the cotyledonary phase (stage IV) of embryogenesis in green cotyledons of seedlings and in young roots. We have characterized two distinct polypeptides with 1-L-myo-inositol-1-phosphate synthase (MI-1-P synthase) activity that are differentially expressed during development in Phaseolus vulgaris. Western analyses, enzyme assays, and partial purification of MI-1-P synthase during embryonic and postembryonic development show that its expression is temporally and spatially regulated. Developmental Western analyses of soluble proteins detect a small protein, approximately 33 kDa, with MI-1-P synthase activity during the globular stage (stage II) of embryogenesis and in mature roots. Expression of this small protein is also enriched in thylakoidal membranes of fractionated leaf chloroplasts, although Western analyses of total soluble leaf proteins show no cross-reacting material. In contrast, a larger protein, approximately 56 kDa, with MI-1-P synthase activity is present during the cotyledonary phase (stage IV) of embryogenesis in green cotyledons of seedlings and in young roots. INTRODUCTIONInositol, a six-carbon cyclitol, is an essential component of eukaryotic cells. Its metabolism is an important concern in many agricultural and clinical disciplines. Inositol phosphates convey signals for a wide variety of hormones, growth factors, and neurotransmitters (1Hokin L.E. Annu. Rev. Biochem. 1985; 54: 205-235Crossref PubMed Scopus (482) Google Scholar, 2Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3297) Google Scholar, 3Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar). In addition, inositol acts as a precursor for a bewildering array of compounds in plant cells (4Drobak B.K. Biochem. J. 1993; 288: 697-712Crossref Scopus (142) Google Scholar).Inositol 1-phosphate, the immediate precursor of free inositol, is synthesized via an internal cyclization of glucose 6-phosphate (5Eisenberg F. Bolden A. Biochem. Biophys. Res. Commun. 1962; 12: 72-77Crossref Scopus (20) Google Scholar, 6Loewus F.A. Kelly S. Biochem. Biophys. Res. Commun. 1962; 7: 204-208Crossref PubMed Scopus (43) Google Scholar, 7Eisenberg Jr., F. Bolden A.H. Loewus F.A. Biochem. Biophys Res. Commun. 1964; 14: 419-424Crossref PubMed Scopus (31) Google Scholar, 8Chen I.W. Charalampous F.C. J. Biol. Chem. 1966; 241: 2194-2199Abstract Full Text PDF PubMed Google Scholar). The enzyme that catalyzes this reaction, MI-1-P synthase, 1The abbreviations used are: MI-1-P synthase1-L-myo-inositol-1-phosphate synthase (EC)HPLChigh performance liquid chromatography. has been purified or partially purified from a number of organisms (9Loewus M.W. Loewus F.A. Plant Physiol. (Bethesda). 1971; 48: 255-260Crossref PubMed Google Scholar, 10Ogunyemi E.O. Pittner F. Hoffman-Ostenhof O. Hoppe-Seyler's Z. Physiol. Chem. 1978; 359: 613-616PubMed Google Scholar, 11Maeda T. Eisenberg F. J. Biol. Chem. 1980; 255: 8458-8464Abstract Full Text PDF PubMed Google Scholar, 12Donahue T.F. Henry S.A. J. Biol. Chem. 1981; 256: 7077-7085Abstract Full Text PDF PubMed Google Scholar). Properties and catalytic mechanisms of MI-1-P synthase are similar in animals, plants, and yeast (14Loewus M.W. Loewus F.A. Plant Sci. Lett. 1973; 1: 65-69Crossref Scopus (5) Google Scholar, 15Kiely D.E. Sherman W.R. J. Am. Chem. Soc. 1975; 97: 6810-6814Crossref PubMed Scopus (18) Google Scholar, 16Sherman W.R. Rasheed A. Mauck L.A. Wiecko J. J. Biol Chem. 1977; 252: 5672-5676Abstract Full Text PDF PubMed Google Scholar, 17Wong Y.-H.H. Sherman W.R. J. Biol. Chem. 1985; 260: 11083-11090Abstract Full Text PDF PubMed Google Scholar, 18Loewus F.A. Boss W.F. Morré D.J. Loewus F.A. Inositol Metabolism in Plants. Wiley-Liss Inc., New York1990: 13Google Scholar). The overall reaction consists of a tightly coupled oxidation and reduction (reviewed in 18Loewus F.A. Boss W.F. Morré D.J. Loewus F.A. Inositol Metabolism in Plants. Wiley-Liss Inc., New York1990: 13Google Scholar).Although extensive physiological and biochemical data document the importance of inositol to higher plants (e.g. seed germination, membrane formation, cell wall biogenesis, and stress response; reviewed in 18Loewus F.A. Boss W.F. Morré D.J. Loewus F.A. Inositol Metabolism in Plants. Wiley-Liss Inc., New York1990: 13Google Scholar), less is known of the molecular genetic mechanisms regulating its metabolism. Our objective is to define genetic controls involved in the regulation of inositol synthesis and catabolism in plants. To this end, we have begun developmental studies of the genes and gene products of the pivotal biosynthetic enzyme, MI-1-P synthase, using the well characterized developmental biology of the green bean, Phaseolus vulgaris, and the molecular and classic genetics available in Arabidopsis thaliana.This article reports intriguing findings concerning the temporal and spatial expression of MI-1-P synthase during development of P. vulgaris.DISCUSSIONOur biochemical analyses of the developmental regulation of MI-1-P synthase expression in the green bean establish, for the first time, the foundation needed to genetically dissect the regulation of inositol metabolism in a higher plant. Phaseolus provides a useful model system in which to study, at the molecular level, the regulation of inositol biosynthesis during plant growth and development. Embryonic and postgerminative development of organs proceed along a predictable pathway. Furthermore, all stages of development provide ample experimental material for biochemical analyses.Although we observed no cross-reacting material when soluble leaf proteins were analyzed by Western blotting, synthase activity in the ammonium sulfate-saturated precipitates of leaf proteins clearly pointed to its presence in leaves. Western analyses of the precipitates showed that the 33-kDa form of the enzyme was indeed located in the leaves. Subsequent isolation and fractionation of Phaseolus leaf chloroplasts localized the 33-kDa form of the enzyme to the thylakoids, confirming previous reports of MI-1-P synthase activity associated with these organelles. We also, surprisingly, identified a very large cross-reacting protein or protein complex (approximately 80 kDa) with MI-1-P synthase activity, which localized to the chloroplast envelope and stroma. This protein could represent an active precursor, since most proteins targeted to the thylakoids are synthesized outside of the chloroplasts as larger molecular size precursors and processed to their mature size during transport (35Viitanen P.V. Doran E.R. Dunsmuir P. J. Biol. Chem. 1988; 263: 15000-15007Abstract Full Text PDF PubMed Google Scholar, 36Lamppa G.K. J. Biol. Chem. 1988; 263: 14996-14999Abstract Full Text PDF PubMed Google Scholar, 37Cai D. Herrmann R.G. Klosgen R.B. Plant J. 1993; 3: 383-392Crossref Scopus (20) Google Scholar). Routine Western analyses and ammonium sulfate fractionation cannot detect this protein.The developmental profile for the expression of MI-1-P synthase in Phaseolus is striking. Expression and associated activity appears at two crucial stages (stages II and IV) during seed maturation. During these stages, two important transient organs, the suspensor (stage II) and cotyledons (stage IV), are formed. Given the postulated functions for the angiosperm suspensor (anchor and/or major route of nutrients into the embryo), it will be of great interest to determine the subcellular localization of MI-1-P synthase (inositol biosynthesis) during this stage of embryogenesis. More importantly, we can now begin to investigate the role of inositol in the developing embryo and/or suspensor. It will also be informative to localize inositol biosynthesis in embryonic and postembryonic cotyledons and to compare the site of inositol synthesis with that of phytic acid (a hexakisphosphoric acid ester of myo-inositol that serves as a major phosphate reserve for the seed).Our working hypothesis based on the data presented and the probability that inositol cannot traverse the plastid membrane (38Wang C.T. Nobel P.S. Biochim. Biophys. Acta. 1971; 241: 200-212Crossref PubMed Scopus (27) Google Scholar) is that: 1) MI-1-P synthase (de novo biosynthesis of inositol) in Phaseolus is localized to plastids in different organs (roots, leaves, suspensor and/or embryo, and cotyledons); and 2) different forms of the enzyme at different stages of development reflect regulatory controls at the transcriptional and translational levels. Support for this hypothesis comes from preliminary Northern analyses, the recent cloning and sequencing of a root cDNA encoding a protein with MI-1-P synthase activity (39Wang X. Johnson M.D. Plant Physiol. (Bethesda). 1995; 110: 336Google Scholar), the recent isolation of a Phaseolus leaf cDNA clone, 2M. D. Johnson and X. Wang, unpublished data. and the presence of an inositol transport system in plants (18Loewus F.A. Boss W.F. Morré D.J. Loewus F.A. Inositol Metabolism in Plants. Wiley-Liss Inc., New York1990: 13Google Scholar).Developmental studies of inositol biosynthesis in Arabidopsis and the isolation of a second Arabidopsis cDNA with MI-1-P synthase activity (31Johnson M.D. Sussex I.M. Plant Physiol. (Bethesda). 1995; 107: 613-619Crossref PubMed Scopus (57) Google Scholar, 40Johnson M.D. Burk D.H. Plant Physiol. (Bethesda). 1995; 109: 721Crossref PubMed Scopus (3) Google Scholar, 41Johnson M.D. Plant Physiol. (Bethesda). 1994; 105: 1023-1024Crossref PubMed Scopus (42) Google Scholar) also suggest that transcriptional and translational mechanisms operate to regulate the temporal and spatial expression of MI-1-P synthase in Arabidopsis.These recent findings and the infinite questions and possibilities raised concerning the genetic regulation of inositol biosynthesis in higher plants propel us into the exciting area of chloroplast (plastid) molecular biology. Future experiments, among many, will address the mechanics of translocation of MI-1-P synthase into the chloroplast. INTRODUCTIONInositol, a six-carbon cyclitol, is an essential component of eukaryotic cells. Its metabolism is an important concern in many agricultural and clinical disciplines. Inositol phosphates convey signals for a wide variety of hormones, growth factors, and neurotransmitters (1Hokin L.E. Annu. Rev. Biochem. 1985; 54: 205-235Crossref PubMed Scopus (482) Google Scholar, 2Berridge M.J. Irvine R.F. Nature. 1989; 341: 197-205Crossref PubMed Scopus (3297) Google Scholar, 3Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6148) Google Scholar). In addition, inositol acts as a precursor for a bewildering array of compounds in plant cells (4Drobak B.K. Biochem. J. 1993; 288: 697-712Crossref Scopus (142) Google Scholar).Inositol 1-phosphate, the immediate precursor of free inositol, is synthesized via an internal cyclization of glucose 6-phosphate (5Eisenberg F. Bolden A. Biochem. Biophys. Res. Commun. 1962; 12: 72-77Crossref Scopus (20) Google Scholar, 6Loewus F.A. Kelly S. Biochem. Biophys. Res. Commun. 1962; 7: 204-208Crossref PubMed Scopus (43) Google Scholar, 7Eisenberg Jr., F. Bolden A.H. Loewus F.A. Biochem. Biophys Res. Commun. 1964; 14: 419-424Crossref PubMed Scopus (31) Google Scholar, 8Chen I.W. Charalampous F.C. J. Biol. Chem. 1966; 241: 2194-2199Abstract Full Text PDF PubMed Google Scholar). The enzyme that catalyzes this reaction, MI-1-P synthase, 1The abbreviations used are: MI-1-P synthase1-L-myo-inositol-1-phosphate synthase (EC)HPLChigh performance liquid chromatography. has been purified or partially purified from a number of organisms (9Loewus M.W. Loewus F.A. Plant Physiol. (Bethesda). 1971; 48: 255-260Crossref PubMed Google Scholar, 10Ogunyemi E.O. Pittner F. Hoffman-Ostenhof O. Hoppe-Seyler's Z. Physiol. Chem. 1978; 359: 613-616PubMed Google Scholar, 11Maeda T. Eisenberg F. J. Biol. Chem. 1980; 255: 8458-8464Abstract Full Text PDF PubMed Google Scholar, 12Donahue T.F. Henry S.A. J. Biol. Chem. 1981; 256: 7077-7085Abstract Full Text PDF PubMed Google Scholar). Properties and catalytic mechanisms of MI-1-P synthase are similar in animals, plants, and yeast (14Loewus M.W. Loewus F.A. Plant Sci. Lett. 1973; 1: 65-69Crossref Scopus (5) Google Scholar, 15Kiely D.E. Sherman W.R. J. Am. Chem. Soc. 1975; 97: 6810-6814Crossref PubMed Scopus (18) Google Scholar, 16Sherman W.R. Rasheed A. Mauck L.A. Wiecko J. J. Biol Chem. 1977; 252: 5672-5676Abstract Full Text PDF PubMed Google Scholar, 17Wong Y.-H.H. Sherman W.R. J. Biol. Chem. 1985; 260: 11083-11090Abstract Full Text PDF PubMed Google Scholar, 18Loewus F.A. Boss W.F. Morré D.J. Loewus F.A. Inositol Metabolism in Plants. Wiley-Liss Inc., New York1990: 13Google Scholar). The overall reaction consists of a tightly coupled oxidation and reduction (reviewed in 18Loewus F.A. Boss W.F. Morré D.J. Loewus F.A. Inositol Metabolism in Plants. Wiley-Liss Inc., New York1990: 13Google Scholar).Although extensive physiological and biochemical data document the importance of inositol to higher plants (e.g. seed germination, membrane formation, cell wall biogenesis, and stress response; reviewed in 18Loewus F.A. Boss W.F. Morré D.J. Loewus F.A. Inositol Metabolism in Plants. Wiley-Liss Inc., New York1990: 13Google Scholar), less is known of the molecular genetic mechanisms regulating its metabolism. Our objective is to define genetic controls involved in the regulation of inositol synthesis and catabolism in plants. To this end, we have begun developmental studies of the genes and gene products of the pivotal biosynthetic enzyme, MI-1-P synthase, using the well characterized developmental biology of the green bean, Phaseolus vulgaris, and the molecular and classic genetics available in Arabidopsis thaliana.This article reports intriguing findings concerning the temporal and spatial expression of MI-1-P synthase during development of P. vulgaris.