Myo-inositol Oxidation Pathway Research Articles

Potential of engineering the myo-inositol oxidation pathway to increase stress resilience in plants.

Myo-inositol is one of the most abundant form of inositol. The myo-inositol (MI) serves as substrate to diverse biosynthesis pathways and hence it is conserved across life forms. The biosynthesis of MI is well studied in animals. Beyond biosynthesis pathway, implications of MI pathway and enzymes hold potential implications in plant physiology and crop improvement. Myo-inositol oxygenase (MIOX) enzyme catabolize MI into D-glucuronic acid (D-GlcUA). The MIOX enzyme family is well studied across few plants. More recently, the MI associated pathway's crosstalk with other important biosynthesis and stress responsive pathways in plants has drawn attention. The overall outcome from different plant species studied so far are very suggestive that MI derivatives and associated pathways could open new directions to explore stress responsive novel metabolic networks. There are evidences for upregulation of MI metabolic pathway genes, specially MIOX under different stress condition. We also found MIOX genes getting differentially expressed according to developmental and stress signals in Arabidopsis and wheat. In this review we try to highlight the missing links and put forward a tailored view over myo-inositol oxidation pathway and MIOX proteins.

Biochemical characterization and spatio-temporal expression of myo-inositol oxygenase (MIOX) from wheat (Triticum aestivum L.)

Myo-inositol oxygenase (MIOX) catalyzes the conversion of myo-inositol into d-glucuronic acid. The present study demonstrates isolation of MIOX cDNA (TaMIOX) from wheat (Triticum aestivum L.) with open reading frame of 912bp encoding 303 amino acid polypeptides with a molecular mass of 35.2kDa. Phylogenetic analysis of TaMIOX across kingdoms confirmed the close relationship with Triticum urartu and Aegilops tauschii. Secondary structure of TaMIOX consists of α-helixes (42.9%), β-turns (7.26%) joined by extended strands (14.85%), and 37 random coils (34.94%). Three-dimensional structure of TaMIOX suggested its close functional and structural resemblance with known MIOX. Catalytic activity of the purified TaMIOX is 3.47μkatal at pH8.0 and 35°C with Michaelis constant 5.6mM. Differential expression pattern of TaMIOX was observed in leaves, root, stem, seed and seed developmental stages. Leaves showed a significantly higher transcript accumulation followed by root, stem and seed. Expression of TaMIOX during seed development stages showed higher expression at later stage and suggested that expression of TaMIOX was significantly higher in endosperm as compared with aleurone. The exogenous application of myo-inositol enhanced the expression of TaMIOX in leaves and roots suggested myo-inositol acts as an inducer for the TaMIOX expression. The present study reports molecular, structural and biochemical characterizations of MIOX in wheat which might play an important role in myo-inositol oxidation pathway.

Arabidopsis UDP-sugar pyrophosphorylase: Evidence for two isoforms

Arabidopsis UDP-sugar pyrophosphorylase (AtUSP, EC 2.7.7.64) is a broad substrate pyrophosphorylase that exhibits activity with GlcA-1-P, Gal-1-P and Glc-1-P. Immunoblots using polyclonal antibodies raised to recombinant AtUSP demonstrated the presence of two USP isoforms of approximately 70 kDa (USP1) and 66 kDa (USP2) in crude extracts of Arabidopsis tissues. The 66 kDa isoform was not the result of proteolytic cleavage of USP1 during extraction. Trypsin digestion of bands on SDS gels corresponding to the location of the two isoforms followed by tandem mass spectrometry confirmed that USP peptides were present in both bands. Both USP isoforms were detected in the cytosol as determined by immunoblots of cellular fractions obtained by differential centrifugation. However, some USP1 was also detected in the microsomal fraction. Immunoprecipitation assays demonstrated that AtUSP antibodies removed USP activity (UDP-GlcA-->GlcA-1-P) measured in floret extracts. These results indicate that USP is the only pyrophosphorylase that utilizes UDP-GlcA as a substrate and suggest that it serves as the terminal enzyme of the myo-inositol oxidation pathway.

Ultrastructure, chemical composition and biosynthesis of the cell wall in Koliella antarctica (Klebsormidiales, Chlorophyta)

The chemical composition, biosynthesis and ultrastructural organization of the cell wall of cultures of Koliella antarctica (Klebsormidiales, Chlorophyta), a green microalga obtained from samples of sea water taken near the Italian Station of Terra Nova (Ross Sea) during the austral summer 1989–1990, have been studied. Purified cell walls of the microalga were sequentially treated with chemicals which, in higher plants, solubilize matrix polysaccharides (pectins and hemicelluloses) and leave an insoluble residue considered to be α-cellulose. CDTA plus Na2CO3- and KOH-solubilized polysaccharides were made up of glucosyl residues (more than 90 mol%), as well as some minor sugars (mannose and rhamnose). Linkage analysis and enzymic hydrolysis of the matrix polysaccharides indicated the presence of β-1,4-linked glucans with minor amounts of other uncharacterized polysaccharides. The insoluble residue was composed of a small amount of crystalline α-cellulose associated with mannan or glucomannan chains. The biosynthesis of cell wall polysaccharides was studied by incubating the microalgae in the presence of D-[U-14C]glucose or myo-[U-14C]inositol. The radioactive glycosyl residues incorporated into the polysaccharides solubilized from purified cell walls of the cultured microalga corresponded to those detected in the chemical analysis of the cell wall. The myo-inositol oxidation pathway was demonstrated to be functional in the microalga. Cell wall ultrastructural observations showed a loose network of cellulose microfibrils in an extended matrix.

Use of Per-C-Deuterated myo-Inositol for Study of Cell Wall Synthesis in Germinating Beans

Cell wall polysaccharides of the hypocotyl and roots in germinating beans (Phaseolus vulgaris L.) were selectively labeled in arabinosyl, xylosyl, and galacturonosyl residues by per-C-deuterated myo-inositol, which was introduced through 72 hours of imbibition. Glucuronate residues remained unlabeled. Selected ion gas chromatography-mass spectrometry analysis revealed that deuterium was not redistributed in these three sugar residues or into other carbohydrate residues during this conversion, suggesting that the labeled residues are formed exclusively via the myo-inositol oxidation pathway and that no glucogenesis from myo-inositol takes place during this conversion. The presence of a significant level of deuterated arabinose, xylose, and galacturonate after just 72 hours of imbibitional uptake of per-C-deuterated myo-inositol indicated that the myo-inositol oxidation pathway has a predominant role in the biosynthesis of new cell walls.

Myo-Inositol Synthesis from [1-3H]Glucose in Phaseolus vulgaris L. during Early Stages of Germination

Radiolabeled d-[1-(3)H]glucose was fed by imbibition under sterile conditions to bean (Phaseolus vulgaris L.) seeds. After 72 and 96 hours of feeding, the (3)H was located in uronic acid and pentose residues as well as hexose residues of cell wall polysaccharides in growing hypocotyl and root. Free myo-inositol present in cotyledons, hypocotyl, and root also contained (3)H, showing that de novo synthesis of myo-inositol from [1-(3)H]glucose did occur during the first 72 hours of germination. More than 90% of the labeled, free myo-inositol was present in the cotyledons. The (3)H percentage in trifluoroacetic acid-soluble arabinose residues of cell wall polysaccharides from 72-hour-old bean hypocotyls was only half of their mole percentage. On the other hand, (3)H percentages in hexose residues were higher than their mole percentages. The results suggest that myo-inositol is synthesized from reserve sugars during the very early stages of germination, and that the newly synthesized myo-inositol, as well as that stored in cotyledons, can be used for the construction of new hypocotyl and root cell wall polysaccharides after conversion into uronic acids and pentoses via the myo-inositol oxidation pathway.

Myo-Inositol-1-Phosphatase from the Pollen of Lilium longiflorum Thunb.

A Mg(2+)-dependent, alkaline phosphatase has been isolated from mature pollen of Lilium longiflorum Thunb., cv. Ace and partially purified. It hydrolyzes 1l- and 1d-myo-inositol 1-phosphate, myo-inositol 2-phosphate, and beta-glycerophosphate at rates decreasing in the order named. The affinity of the enzyme for 1l- and 1d-myo-inositol 1-phosphate is approximately 10-fold greater than its affinity for myo-inositol 2-phosphate. Little or no activity is found with phytate, d-glucose 6-phosphate, d-glucose 1-phosphate, d-fructose 1-phosphate, d-fructose 6-phosphate, d-mannose 6-phosphate, or p-nitrophenyl phosphate. 3-Phosphosphoglycerate is a weak competitive inhibitor. myo-Inositol does not inhibit the reaction. Optimal activity is obtained at pH 8.5 and requires the presence of Mg(2+). At 4 millimolar, Co(2+), Fe(2+) or Mn(2+) are less effective. Substantial inhibition is obtained with 0.25 molar Li(+). With beta-glycerophosphate as substrate the K(m) is 0.06 millimolar and the reaction remains linear at least 2 hours. In 0.1 molar Tris, beta-glycerophosphate yields equivalent amounts of glycerol and inorganic phosphate, evidence that transphosphorylation does not occur.In higher plants this myo-inositol-1-phosphatase links myo-inositol biosynthesis to the myo-inositol oxidation pathway to produce an alternative path from d-glucose 6-phosphate to UDP-d-glucuronate that bypasses UDP-d-glucose dehydrogenase. myo-Inositol-1-phosphatase also furnishes free myo-inositol for reactions that lead to other cyclitols and cyclitol-containing compounds of biosynthetic and/or regulatory significance in plant growth and development.

Redistribution of Tritium during Germination of Grain Harvested from myo-[2-3H]Inositol- and scyllo-[R-3H]Inositol-Labeled Wheat

Wheat kernels from myo-[2-(3)H]inositol- or scyllo-[R-(3)H]inositol-labeled plants (Sasaki and Loewus 1980 Plant Physiol 66: 740-745) were used to study redistribution of (3)H into growing regions during germination. Most of the labeled 1-alpha-galactinol (or the analogous scyllo-inositol galactoside) was hydrolyzed within 1 day. Water-soluble phytate was dephosphorylated within 3 days. A large reserve of bound phytate continued to release myo-inositol over several days. Translocation of free myo-inositol to growing regions provided substrate for the myo-inositol oxidation pathway and incorporation of (3)H into new cell wall polysaccharides.Cell wall polysaccharides in the kernel were degraded during germination. The labeled residues were translocated to growing regions and reutilized for new cell wall formation. Pentosyl residues accounted for most of this label.Free scyllo-inositol followed a path of translocation from kernel to seedling similar to that of myo-inositol. Unlike myo-inositol, it did not furnish substrate for the myo-inositol oxidation pathway but accumulated as free scyllo-inositol in the seedling.The fate of phytate-derived myo-inositol during germination of wheat is discussed in relation to a recent scheme of phytate metabolism proposed by De and Biswas (1979 J Biol Chem 254: 8717-8719) for germinating mung bean seedlings.

The C-5 hydrogen isotope-effect in myo-inositol 1-phosphate synthase as evidence for the myo-inositol oxidation-pathway

The hydrogen isotope-effect that occurs in vitro during myo-inositol 1-phosphate synthase-catalyzed conversion of d-[5- 3H]glucose 6-phosphate into myo-[2- 3H]inositol 1-phosphate has been used to compare the functional role of the nucleotide sugar oxidation-pathway with that of the myo-inositol oxidation-pathway in germinating lily pollen. Results reveal a significant difference between the 3H/ 14C ratios of glucosyl and galactosyluronic residues from pectinase-amyloglucosidase hydrolyzates of the 70 % ethanol-insoluble fraction of d-[5- 3H, 1- 14-C]glucose-labeled, germinating lily pollen. This isotope effect at C-5 of d-glucose that occurred during its conversion into d-galactosyluronic residues of pectic substance is not explained by loss of 3H when UDP- d-[5- 3H, 1- 14C]glucose is oxidized by UDP- d-glucose dehydrogenase from germinating lily pollen. The evidence obtained from this study favors a functional role for the myo-inositol oxidation pathway during in vivo conversion of glucose into galactosyluronic residues of pectin in germinating lily pollen.

Labeling and chemistry of grapefruit pectic substances

Abstract The labeling of a number of polysaccharides found in grapefruit ( Citrus paradisi ) was achieved by feeding labeled myo-inositol to ripening grapefruit through their cut fruit stem, and allowing 4 days for the metabolism of label. The pectic polysaccharides were isolated by successive extraction of the labeled grapefruit with 80% ethanol, chloroform-methanol (1:1) and finally with 0.2 M Na 2 EDTA to solubilize pectic polysaccharides. The incorporation of label from myo-inositol into galacturonosyl, arabinosyl, xylosyl and galactosyl residues of pectic polysaccharides via myo-inositol oxidation pathway was demonstrated. Ion exchange chromatography of these labeled pectic polysaccharides using DE-52 cellulose resulted in the elution of eight totally or partially resolved polysaccharides with increasing salt concentration. The results suggest that, like other plant tissues, the myo-inositol oxidation pathway is also operative in ripening grapefruit and this metabolic pathway could be successfully utilized to achieve labeling of a number of pectic polysaccharides.

Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-(14)C]glucose and [2-(3)H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [(14)C]MI from [U-(14)C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of (3)H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No (3)H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen

The myo-inositol oxidation pathway was investigated in regard to its role as a source of carbon for products of hexose monophosphate metabolism in germinated pollen of Lilium longiflorum Thunb., cv. Ace. myo-[2-(14)]Inositol and d-[1-(14)C]glucuronate had similar distributions of radioactivity, contributing about three times more label to polysaccharide-bound glucose than myo-[2-(3)H]inositol. In the course of glucogenesis label from the latter appeared as tritiated water in the medium. This exchange could be enhanced by supplying d-[5R,5S-(3)H]xylose instead of myo-[2-(3)H]inositol. When the former was administered, [(3)H]glucose was the only labeled sugar residue found in polysaccharide products. The soluble constituents of d-[5R,5S-(3)H]xylose-labeled pollen contained no traces of labeled xylose despite massive uptake and utilization.l-[1-(14)C]- and l-[5-(14)C]Arabinose produced similar labeling patterns in germinated pollen including incorporation of arabinosyl units into pollen tube polysaccharides and substantial glucogenesis which led to utilization of arabinose for respiration and further incorporation of labeled glucosyl units into pollen tube polysaccharides.d-[5-(3)H]Galacturonate was rapidly taken up by germinated pollen but slowly utilized, without conversion to other sugars, for incorporation into pollen tube polysaccharides. l-[6-(14)C]Gulonate was not taken up by pollen.Results strongly support a scheme of conversion from myo-inositol to hexose monophosphate and subsequent products of glucose metabolism that involves the myo-inositol oxidation pathway.

Myo-Inositol metabolism in germinating wheat

During imbibition, exogenous myo-inositol (MI) was readily introduced into the free MI pool of germinating wheat (Triticum aestivum L.). Maximum uptake, 70 μg per caryopsis or 1.5 mg g(-1) of caryopsis, was reached at 0.05 M MI. Movement of free MI within the germinating caryopsis was traced with [2-(3)H]MI by two procedures, uptake by imbibition and injection into softened endosperm. The former procedure was useful during initial stages of germination; the latter provided a means of tracing the metabolic fate of MI generated by hydrolysis of phytate during mobilization of reserves within the caryopsis. In both procedures, the bulk of the added label was transferred to the seedling where it appeared in uronosyl and pentosyl units of 80% ethanol-insoluble polysaccharides, 2-O, C-Methylene-MI, an inhibitor of the MI oxidation pathway, blocked the utilization of [2-(3)H]MI as well as D-[1(14)C]glucose for biogenesis of pentose-and uronic-acid-containing polysaccharides.

Metabolic Studies on Intermediates in the myo-Inositol Oxidation Pathway in Lilium longiflorum Pollen

myo-Inositol-linked glucogenesis in germinated lily (Lilium longiflorum Thunb., cv. Ace) pollen was investigated by studying the effects of added l-arabinose or d-xylose on metabolism of myo-[2-(3)H]inositol and by determining the distribution of radioisotope in pentosyl and hexosyl residues of polysaccharides from pollen labeled with myo-[2-(14)C]inositol, myo-[2-(3)H]inositol, l-[5-(14)C]arabinose, and d-[5R,5S-(3)H]xylose.myo-[2-(14)C]Inositol and l-[5-(14)C]arabinose produced labeled glucose with similar patterns of distribution of (14)C, 35% in C1, and 55% in C6. Arabinosyl units were labeled exclusively in C5. Incorporation of (3)H into arabinosyl and xylosyl units in pollen labeled with myo-[2-(3)H]inositol was repressed when unlabeled l-arabinose was included in the germination medium and a related (3)H exchange with water was stimulated. Results are consistent with a process of glucogenesis in which the myo-inositol oxidation pathway furnishes UDP-d-xylose as a key intermediate for conversion to hexose via free d-xylose and the pentose phosphate pathway.Additional evidence for this process was obtained from pollen labeled with d-[5R,5S-(3)H]xylose or myo-[2-(3)H]inositol which produces d-[5R-(3)H]xylose. Glucosyl units from polysaccharides in the former had 11% of the (3)H in C1 and 78% in C6 while glucosyl units in the latter had only 4% in C1 and 78% in C6. Stereochemical considerations involving selective exchange with water of prochiral-R (3)H in C1 of fructose-6-P during conversion to glucose provide explanation for observed differences in the metabolism of these 5-labeled xyloses.Incorporation of (3)H from myo-[2-(3)H]inositol into arabinosyl and xylosyl units of pollen polysaccharides was unaffected by the presence of unlabeled d-xylose in the medium. Exchange of (3)H with water was greatly affected, decreasing from a value of 21% exchange in the absence of unlabeled d-xylose to 5% in the presence of 6.7 mmd-xylose.d-Xylose was rapidly utilized for glucogenesis by germinated pollen tubes. This observation supports the view that free d-xylose is an important intermediate following breakdown of UDP-d-xylose during myo-inositol-linked glucogenesis.

Purification of myo-Inositol 1-Phosphate Synthase from Rice Cell Culture by Affinity Chromatography

myo-Inositol 1-phosphate synthase (EC 5.5.1.4) is the enzyme which catalyzes the synthesis of the precursor for the myo-inositol oxidation pathway. Rice callus grown in suspension culture provides a good source of plant enzyme. Use has been made of a noncompetitive inhibitor to prepare an affinity column for this enzyme. With this column, the enzyme from rice callus has been purified 1500-fold in a single step, about 9000-fold over-all, to a specific activity of 0.078 units per milligram of protein. This is an order of magnitude greater than previous purifications of the plant enzyme.